&#34;Methods and compositions for decreasing the frequency of HIV, herpesvirus and sexually transmitted bacterial infections&#34;

ABSTRACT

A method for decreasing the frequency of transmission of human immunodeficiency virus or herpesviruses or for preventing the transmission of or treating a sexually transmitted bacterial infection by administering to a human an anti-human immunodeficiency virus amount or an anti-herpesvirus amount or an anti-bacterial amount of cellulose acetate phthalate (CAP) or hydroxypropyl methylcellulose phthalate (HPMCP), such as in micronized form, or a combination thereof, either alone or in combination with a pharmaceutically acceptable carrier or diluent. The CAP and/or HPMCP may be employed as a suspension of micronized particles and may further contain a water miscible, non-solvent for CAP or HPMCP, such as glycerol.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application ofapplication Ser. No. 09/112,130 filed Jul. 8, 1998, now U.S. Pat. No.5,985,313 wherein priority under 35 USC 119 (e) is claimed forprovisional application Nos. 60/062,936 filed Oct. 22, 1997 and60/071,017, filed Jan. 13, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns methods for decreasing the frequency oftransmission of viral infection, such as human immunodeficiency virusand herpesvirus, and for preventing and treating sexually transmittedbacterial infections, such as Chlamydia trachomatis, by administrationof cellulose acetate phthalate or hydroxypropyl methylcellulosephthalate, which were heretofore employed as pharmaceutical excipients.

2. Background Information

a. Pharmaceutical Excipients

Pharmaceutical excipients are defined as inert substances that form avehicle for drug delivery (Webster's Ninth New Collegiate Dictionary,Merriam-Webster Inc. Publishers, Springfield, Mass., USA, 1985, p. 432).Thus, excipients convert pharmacologically active compounds intopharmaceutical dosage forms suitable for administration to patients.Some excipients are also used for the formulation or production ofconfectionery, cosmetics and food products. Therefore, approvedexcipients are used frequently and at higher dosage levels in comparisonwith most drugs. Excipients are also much less expensive and more easilyproduced in very large scale in comparison with most drugs.

b. Sexually Transmitted Diseases ("STDs"): An Overview

The human immunodeficiency virus (HIV) pandemic is sustained andprogressing predominantly due to sexual transmission of the virus (Mann,J.. M., Tarantola, D. J. M., Netter, T. W., "AIDS in the World",Cambridge: Harvard University Press, (1992)), facilitated by priorinfection with other STD pathogens (Perine, P. L., "Sexually TransmittedDiseases in the Tropics", Med. J. Aust., 160, (1994), 358-366).

The urgent need to prevent the transmission of STDs has becomehighlighted by the HIV/AIDS epidemic, resulting so far in infection ofapproximately 42 million people and in approximately 12 million deaths(UNAIDS and WHO, Report on the global HIV/AIDS epidemic, Geneva: JointUnited Nations Programme on HIV/AIDS, Jun. 1, 1998). The facts that HIVinfections are not curable as of now, have become the leading cause ofdeath among young adults and has decreased life expectancy in a numberof countries, and the observation that several non-viral STDs facilitateHIV infection, have further emphasized the pressing need for newpreventive approaches.

Treatment of STDs (other than HIV) was found to be a feasible andeconomically justifiable approach to decreasing the rate of HIV-1transmission (St. Louis, M. E., Levine, W. C., Wasserheit, J. N. et al,"HIV Prevention Through Early Detection and Treatment of Other SexuallyTransmitted Diseases--United States Recommendation of the AdvisoryCommittee for HIV and STD Prevention", Mor. Mort. Wkly. Rep., (1998), 47(No. RR-12), 1-24; Over, M., Piot, P., "Human Immunodeficiency VirusInfection and Other Sexually Transmitted Diseases in DevelopingCountries: Public Health Importance and Priorities for ResourceAllocation", J. Infect. Dis., (1996), 174 (Suppl. 2), 162-175). However,this beneficial approach is not sufficient to control the spread ofSTDs, including HIV-1.

In the absence of prophylactic vaccines against STD pathogens and HIV inthe foreseeable future, and of safe anti-HIV-1 drugs affordable indeveloping countries, other simple methods to control the sexualtransmission of STDs, including HIV-1, must be applied. This includesmechanical (condom) and chemical barrier methods and combinationsthereof. Formulations of spermicides shown in vitro to inactivate STDpathogens have been considered for this purpose, but based on theoutcome of clinical safety and efficacy trials, their utility remains indoubt.

The use of chemical barrier methods (topical "microbicides") under thecontrol of women has been proposed as a method to control the sexualtransmission of HIV-1 and other STDs (Alexander, N. J., "Barriers toSexually Transmitted Diseases", Scientific American & Medicine, (1996),3:32-41). The fastest way to introduce topical microbicides intopractice appeared to be the application of over-the-counter (OTC)contraceptives containing the detergent nonoxynol-9 (N-9). N-9 was shownto inactivate in vitro HIV-1 (Hicks, D. R., Martin, L. S., Getchell, J.P. et al., "Inactivation of HTLV-III/LAV-infected Cultures of NormalHuman Lymphocytes by Nonoxynol-9 in vitro", Lancet, (1985), 2:1422-1423;Jennings, R., Clegg, A., "The Inhibitory Effect of Spermicidal Agents onReplication of HSV-2 and HIV-1 in vitro", J. Antimicrob. Chemother.,(1993), 32:71-82), HSV-2 (Sugarman, B., Mummaw, N., "Effects ofAntimicrobial Agents on Growth and Chemotaxis of Trichomonas Vaginalis",Antimicrob. Agents Chemother., (1988), 32:1323-1326) and Chlamydiatrachomatis (Lyons, J. M., Ito, J. I., Jr., "Reducing the Risk ofChlamydia Trachomatis Genital Tract Infection by Evaluating TheProphylactic Potential of Vaginally Applied Chemicals", Clin. Infect.Dis., (1995), 21 (Suppl. 2): S174-S177). N-9 was also found to becytotoxic. This approach seems to be hampered by clinical datasuggesting the adverse effects of some N-9 formulations (Stafford, M.K., Ward, H., Flanagan, A., et al., "Safety Study of Nonoxynol-9 As AVaginal Microbicide: Evidence of Adverse Effects", J. Acquir. Imm.Defic. Synd. Hum. Retrovir., (1998), 17:327-331; Rosenstein, I. J.,Stafford, M. K., Kitchen, V. S. et al., "Effects on Normal Vaginal Floraof Three Intravaginal Microbicidal Agents Potentially Active AgainstHuman Immunodeficiency Virus Type 1", J. Infect. Dis., (1998),177:1386-1390; Kilmarx, P. H., Limpakarnjanarat, K., Supawitkul, S. etal., "Mucosal Disruption Due To Use of A Widely-distributed CommercialVaginal Product: Potential to Facilitate HIV Transmission", AIDS,(1998), 12:767-773) and lack of efficacy in decreasing the rate ofheterosexual HIV-1, gonorrhea and chlamydia transmission by one of theN-9 formulations (Roddy, R. E., Zekeng, L., Ryan, K. A. et al., "AControlled Trial of Nonoxynol 9 Film to Reduce Male-to-FemaleTransmission of Sexually Transmitted Diseases", N. Engl. J. Med.,(1998), 339, 504-510). This indicates that other microbicidal compoundshave to be tested as prophylactic agents against HIV-1 and other STDs.

Considering the urgency in controlling the HIV epidemic, the rapiddevelopment of other microbiocidal formulations is needed. Theintroduction of such microbicide formulations into practice would besignificantly enhanced by using active ingredients with an alreadyestablshed safety record for human use.

To search for safe microbicide formulations, criteria distinct fromthose applied to screening for anti-HIV-1 drugs have to be used, raisingthe possibility that promising microbicides with anti-HIV-1 activity mayheretofore have been missed during extensive screening for therapeuticanti-HIV-1 compounds. The criteria for selection of anti-HIV-1microbicides, as compared with those for therapeutic anti-HIV drugs, canbe summarized as follows: (a) undesirability of systemic spread leadingto preferred consideration of high molecular weight compounds (M_(w) ≧2kD), which are active selectively at the site of application; (b) highdegree of safety and lack of side effects (due to repeated use byhealthy people -as compared with the use of therapeutic anti-HIV-1 drugsby already infected individuals), the safety being augmented by the lackof systemic spread; (c) consideration of compounds with lower specificantiviral activity, which can be compensated for by higherconcentrations of the compounds with established safety, and (d)activity directed to early steps in injection and, preferably, directpathogen inactivation, as implied by the term "microbicide".

Compounds meeting at least some of these criteria are as follows: (a)sulfated polysaccharides (Javan, C. M., Gooderham, N. J., Edwards, R. J.et al., "Anti-HIV Type 1 Activity of Sulfated Derivatives of DextrinAgainst Primary Viral Isolates of HIV Type 1 in Lymphocytes andMonocyte-Derived Macrophages", AIDS Res. Human Retroviruses, (1997), 13,875-880; Stafford, M. K., Cain, D., Rosenstein, I., et al., "APlacebo-Controlled, Double Blind Prospective Study in Healthy FemaleVolunteers of Dextrin Sulphate Gel: A Novel Potential IntravaginalVirucide", J. Acquir. Immune Defic. Syndr. Hum. Retrovirol., (1997), 14,213-218; Zacharopoulos, V. R., Phillips, D. M., "Vaginal Formulations ofCarrageenan Protect Mice From Herpes Simplex Virus Infection", Clin.Diagn. Lab. Immunol., (1997), 4, 465-468; Carlucci, M. J., Pujol, C. A.,Ciancia, M. et al., "Antiherpetic and Anticoagulant Properties ofCarrageenans From the Red Seaweed Gigartina Skottsbergii and TheirCyclized Derivatives: Correlation Between Structure and BiologicalActivity", Int. J. Biol. Macromol., (1997), 20, 97-105) and othersulfonated polymers (however, the virucidal and bacterial activity ofthese compounds has not been established and their activity is ascribedto their ability to interact with target cells to inhibit virus entry(Rusconi, S., Moonis, M., Merill, D. P. et al., "Naphthalene SulfonatePolymers With CD-4-Blocking and Anti-Human Immunodeficiency Virus Type 1Activities", Anticrob. Agents Chemother., (1996), 40, 234-236; McClure,M. O., Moore, J. P., Blanc, D. F. et al., "Investigations into theMechanism By Which Sulfated Polysaccharides Inhibit HIV Infection InVitro", AIDS Res. Hum. Retroviruses, (1992), 8, 19-26); and (b)protegrins which have broad spectrum activity against bacteria andenveloped viruses (Tamamura, H., Murakami, T., Horiuchi, S. et al.,"Synthesis of Protegrin-Related Peptides and Their Antibacterial andAnti-Human Immunodeficiency Virus Activity", Chem. Pharm. Bull.,(Tokyo), (1995), 43, 853-858; Lehrer, R. I., Ganz, T., "EndogenousVertebrate Antibiotics, Defensins, Protegrins, and Other Cysteine-RichAntimicrobial Peptides", Ann. N.Y., Acad. Sci., (1996), 797, 228-239;Qu, X. D., Harwig, S. S., Shafer, W. M. et al., "Protegrin Structure andActivity Against Neisseria Gonorrhoeae", Infect. Immun., (1997), 65,636-639), but they also have undesirable activity against Lactobacilliand their application may have economical disadvantages, as comparedwith that of sulfated polymers.

Since efficacious topical "microbicides" would be expected to be usedrepeatedly over decades, they should have an established safety recordand should preferably not be spread systemically after topicalapplication. They should have the following characteristics: (a) beinexpensive, (b) be produced from widely available resources, (c) have abroad specificity resulting in prevention of transmission of severalSTDs, and (d) inactivate the infectivity of the respective STDpathogens. In accordance with these requirements, some of the presentinventors recently developed a potent anti-HIV and anti-herpesvirusagent, suitable for incorporation into topical gels/creams (Neurath, A.R., Jiang, S., Strick, N. et al., "Bovine β-Lactoglobulin Modified by3-Hydroxyphthalic Anhydride Blocks the CD4 Cell Receptor for HIV",Nature Med., (1996), 2, 230-234; Neurath, A. R., Debnath, A. K., Strick,N. et al., "3-Hydroxyphthaloyl β-Lactoglobulin, I, Optimization ofProduction and Comparison With Other Compounds Considered forChemoprophylaxis of Mucosally Transmitted Human Immunodeficiency VirusType 1", Antiviral Chem. Chemother., (1997), 8, 131-140; Neurath, A. R.,Debnath, A. K. Strick N. et al., "3-Hydroxyphthaloyl β-Lactoglobulin,II, Anti-Human Immunodeficiency Virus Type 1 Activity in in vitroEnvironments Relevant to Prevention of Sexual Transmission of theVirus", Antiviral Chem. Chemother., (1997), 8, 141-148; Neurath, A. R.,Strick, N., Li, Y-Y, "3-Hydroxyphthaloyl β-lactoglobulin, III. AntiviralActivity Against Herpesviruses", Antiviral Chem. Chemother., (1998), 9,177-184; Kokuba, H., Aurelian, L., Neurath, A. R., "3-Hydroxyphthaloylβ-Lactoglobulin, IV, Antiviral Activity in the Mouse Model of GenitalHerpevirus Infection", Antiviral Chem. Chemother., (1998), 9, 353-357,by chemical modification of the bovine milk product β-lactoglobulin with3-hydroxyphthalic anhydride. Possible disadvantages of this antiviralcompound has been the lack of activity against bacterial STD pathogens.

c. Viral Infections

Human immunodeficiency viruses ("HIV") have been known as the causativevirus for AIDS (Acquired Immunodeficiency Syndrome). The prevalence ofAIDS cases is presently increasing at an alarming rate.

Two related retroviruses that can cause AIDS are human immunodeficiencyvirus type 1 (HIV-1) and type 2 (HIV-2). The genomes of these twoviruses are about 50% homologous at the nucleotide level, contain thesame complement of genes, and appear to attack and kill the same humancells by the same mechanism.

HIV-1 was identified in 1983. Virtually all AIDS cases in the UnitedStates are associated with HIV-1 infection. HIV-2 was isolated in 1986from West African AIDS patients.

HIV-1 and HIV-2 are retroviruses in which the genetic material is RNA,rather than DNA. The HIV-1 and HIV-2 viruses carry with them apolymerase (reverse transcriptase) that catalyzes transcription of viralRNA into double-helical DNA.

The viral DNA can exist as an unintegrated form in the infected cell orbe integrated into the genome of the host cell. As presently understood,the HIV enters the T4 lymphocyte where it loses its outer envelope,releasing viral RNA and reverse transcriptase.

The reverse transcriptase catalyzes synthesis of a complementary DNAstrand from the viral RNA template. The DNA helix then inserts into thehost genome where it is known as the provirus. The integrated DNA maypersist as a latent infection characterized by little or no productionof virus or helper/inducer cell death for an indefinite period of time.When the viral DNA is transcribed and translated by the infectedlymphocyte, new viral RNA and proteins are produced to form new virusesthat bud from the cell membrane and infect other cells.

Attempts to treat AIDS with drugs which inhibit reverse transcriptasesuch as 3'-azido-3'-deoxythymidine (AZT) have not been met with adesirable degree of success. Moreover, there is a potential for toxicitywith the use of anti-viral drugs. Thus there is a need for an effectiveand safe means to prevent and treat AIDS.

HIV infections are transmitted by means such as contaminated intravenousdrug needles and through sexual contact. Sexual transmission is the mostfrequent (86%) route of adult HIV-1 infections worldwide (AIDS in theWorld, Harvard University Press, Cambridge, Mass., (1992)).

The transmission of HIV by heterosexual sex poses an especially severeproblem for women. By the year 2,000, it is estimated that 90% of HIVinfections will be acquired via heterosexual intercourse.

The utilization of condoms provides a substantial degree of protectionagainst transmission of HIV and herpesvirus infections during sexualintercourse, but a difficulty arises when condoms are not employed.Moreover, the use of condoms appears to be a culturally and sociallyunacceptable practice in many countries.

Although men can protect themselves from sexually transmitted HIV andherpesvirus infection by using condoms, women who are sexually activehave no similar means. Women can encourage their male sex partners touse a condom, but may not succeed. The female condom, which is justbecoming available, is expensive and there is presently no evidence thatit prevents sexual transmission of HIV or herpesvirus.

Even maintaining a monogamous sexual relationship is no guarantee ofsafety, for if a woman's male partner becomes infected, he can pass thevirus to her. And as more women are infected, so are more babies.

There is presently frustration in the medical field by the bleakprospect for an effective AIDS vaccine in the near future and the severelimitations of drugs that effectively and safely combat HIV.

Due to the present absence of a prophylactic anti-HIV vaccine andbecause of limitations of educational programs, other preventive methodshave been sought. Spermicides with virucidal properties have beenconsidered for this purpose, but their application is contraindicated byadverse effects (Bird, K. D., "The Use of Spermicide ContainingNonoxynol-9 in the Prevention of HIV Infection", AIDS, 5, 791-796(1991)).

Anti-HIV drugs currently in use or expected to be clinically applied inthe near future (Steele, F., "AIDS Drugs Lurch Towards Market", NatureMedicine, 1, 285-286 (1995)) are mostly not targeted to the earlieststeps in the virus replicative cycle, lead to the emergence of drugresistant mutants, and are expensive, suggesting that their applicationfor wide use in topical chemoprophylaxis is unlikely.

Cells which are the primary targets for sexual and mucosal transmissionof HIV, either in the form of free virus or virus-infected cells, havenot been fully defined and may be diverse (Miller, C. J. et al.,"Genital Mucosal Transmission of Simian Immunodeficiency Virus: AnimalModel for Heterosexual Transmission of Human Immunodeficiency Virus", J.Virol., 63, 4277-4284 (1989); Phillips, D. M. and Bourinbaiar, A. S.,"Mechanism of HIV Spread from Lymphocytes to Epithelia", Virology, 186,261-273 (1992); Phillips, D. M., Tan, X., Pearce-Pratt, R. andZacharopoulos, V. R., "An Assay for HIV Infection of Cultured HumanCervix-derived Cells", J. Virol. Methods, 52, 1-13 (1995); Ho, J. L. etal., "Neutrophils from Human Immunodeficiency Virus (HIV)-SeronegativeDonors Induce HIV Replication from HIV-infected Patients MononuclearCells and Cell lines": An In Vitro Model of HIV Transmission Facilitatedby Chlamydia Trachomatis.," J. Exp. Med., 181, 1493-1505 (1995); andBraathen, L. R. & Mork, C. in "HIV infection of Skin Langerhans Cells",In: Skin Langerhans (dendritic) cells in virus infections and AIDS (ed.Becker, Y.) 131-139 (Kluwer Academic Publishers, Boston, (1991)). Suchcells include T lymphocytes, monocytes/macrophages and dendritic cells,suggesting that CD4 cell receptors are engaged in the process of virustransmission (Parr, M. B. and Parr, E. L., "Langerhans Cells and Tlymphocyte Subsets in the Murine Vagina and Cervix", Biology ofReproduction, 44, 491-498 (1991); Pope, M. et al., "Conjugates ofDendritic Cells and Memory T Lymphocytes from Skin Facilitate ProductiveInfection With HIV-1", Cell, 78, 389-398 (1994); and Wira, C. R. andRossoll, R. M., "Antigen-presenting Cells in the Female ReproductiveTract: Influence of Sex Hormones on Antigen Presentation in the Vagina",Immunology, 84, 505-508 (1995)).

Therefore agents blocking HIV-CD4 binding are expected to diminish orprevent virus transmission. Soluble recombinant CD4 cannot be consideredfor this purpose since high concentrations are required to neutralizethe infectivity of primary HIV isolates (Daar, E. S., Li, X. L.,Moudgil, T. and Ho, D. D., "High Concentrations of Recombinant SolubleCD4 are Required to Neutralize Primary Human Immunodeficiency Virus Type1 Isolates", Proc. Natl. Acad. Sci. U.S.A., 87, 6574-6578 (1990), and inthe case of SIV, the infectivity is enhanced by CD4 (Werner, A.,Winskowsky, G. and Kurth, R., "Soluble CD4 Enhances SimianImmunodeficiency Virus SIVagm Infection", J. Virol., 64, 6252-6256(1990)). However, anti-CD4 antibodies are expected to prevent virustransmission independently of subtype and variability, but theirapplication would be too costly (Daar et al, supra, Watanabe, M.,Boyson, J. E., Lord, C. I. and Letvin, N. L. "Chimpanzees Immunized withRecombinant Soluble CD4 Develop Anti-self CD4 Antibody Responses withAnti-human Immunodeficiency Virus Activity", Proc. Natl. Acad. Sci.U.S.A., 89, 5103-5107 (1992); and Perno, C.-F., Baseler, M. W., Broder,S. and Yarchoan, R., "Infection of Monocytes by Human ImmunodeficiencyVirus Type 1 Blocked by Inhibitors of CD4-gp120 Binding, Even in thePresence of Enhancing Antibodies", J. Exp. Med., 171, 1043-1056 (1990)).

There is a need for a safe and effective substance that can be insertedinto the vagina by a foam, gel, sponge or other form to prevent HIV-1 orHIV-2 from infecting cells in the body. It is hoped that such substancebe used by a woman without her partner's knowledge.

Prospects for the near and possibly not so near future to prevent HIV-1transmission by vaccination do not seem good. A recent report thatvaccination with inactivated SIV did not protect African Green monkeysagainst infection with the homologous virus notwithstanding a strongimmune response to SIV does not appear to be encouraging in this respect(Siegel, F., Kurth, R., and Norley, S., (1995), "Neither WholeInactivated Virus Immunogen nor Passive Immunoglobulin Transfer ProtectsAgainst SIV_(agm) Infection in the African Green Monkey Natural Host",J. AIDS, 8, 217-226). Considering this problem, emphasis has been put onattempts to build a chemical barrier to HIV-1 transmission (Taylor,(1994), "Building a Chemical Barrier to HIV-1 Transmission", J. NIHRes., 6, 26-27).

The development of topically applied microbicides, expected to preventsexual (mucosal) transmission of HIV-1, was suggested to need to be"effective against all sexually transmitted diseases and should not beseen, smelled, or felt while in use." It should also be inexpensive andwidely available, and $25 million was expected to be devoted to itsdevelopment in the United States in 1995 (Taylor, (1994) supra).Detergents (nonoxynol-9) as a universal pathogen killer have beenselected for clinical trials. However, not surprisingly, this compoundproved to be deleterious to the host.

Targeting the chemical barrier to transmission of individual pathogenscould perhaps facilitate the development of compounds preventing thetransmission of human immunodeficiency viruses. For example, effectiveblockade of receptors for the viruses might accomplish this goal. Thisconcept may be supported by the finding that immunization of chimpanzeesand rhesus monkeys, respectively, with human CD4 which has several aminoacid point mutations in comparison with non-human primate CD4 sequences(Fomsgaard, A., Hirsch, V. M., and Johnson, P. R., (1992), "Cloning andSequences of Primate CD4 molecules: Diversity of the Cellular Receptorfor Simian Immunodeficiency Virus/Human Immunodeficiency Virus", Eur. J.Immunol., 22, 2973-2981), developed anti-CD4 antibodies which inhibitedHIV-1 and SIV replication (Watanabe, M., Levine, C. G., Shen, L.,Fisher, R. A., and Letvin, N. L. (1991), "Immunization of SimianImmunodeficiency Virus-Infected Rhesus Monkeys with Soluble Human CD4Elicits an Antiviral Response," Proc. Natl. Acad. Sci. USA, 88,4616-4620. Watanabe, M., Chen, Z. W., Tsubota, H., Lord, C. I., Levine,C. G., and Letvin, N. L., (1991), "Soluble Human CD4 Elicits an AntibodyResponse in Rhesus Monkeys that Inhibits Simian Immunodeficiency VirusReplication", Proc. Natl. Acad. Sci. USA, 88, 120-124; and Watanabe, M.,Boyson, J. E., Lord, C. I., and Letvin, N. L., (1992), "ChimpanzeesImmunized with Recombinant Soluble CD4 Develop Anti-self CD4 AntibodyResponses with Anti-human Immunodeficiency Virus Activity", Proc. Natl.Acad. Sci. USA, 89, 5103-5107).

Herpesviruses include the following viruses isolated from humans:

(1) herpes simplex virus 1 ("HSV-1")

(2) herpes simplex virus 2 ("HSV-2")

(3) human cytomegalovirus ("HCMV")

(4) varicella-zoster virus ("VZV")

(5) Epstein-Barr virus ("EBV")

(6) human herpesvirus 6 ("HHV6")

(7) herpes simplex virus 7 ("HSV-7")

(8) herpes simplex virus 8 ("HSV-8")

Herpesviruses have also been isolated from horses, cattle, pigs(pseudorabies virus ("PSV") and porcine cytomegalovirus), chickens(infectious larygotracheitis), chimpanzees, birds (Marck's diseaseherpesvirus 1 and 2), turkeys and fish (see "Herpesviridae: A BriefIntroduction", Virology, Second Edition, edited by B. N. Fields, Chapter64, 1787 (1990)).

Herpes simplex viral ("HSV") infection is generally a recurrent viralinfection characterized by the appearance on the skin or mucousmembranes of single or multiple clusters of small vesicles, filled withclear fluid, on slightly raised inflammatory bases.

The herpes simplex virus is a relatively large-sized virus. HSV-2commonly causes herpes labialis. HSV-2 is usually, though not always,recoverable from genital lesions. Ordinarily, HSV-2 is transmittedvenereally.

At least 20% of people in the United States have been infected withherpesvirus type 2 (HSV-2), which is usually transmitted sexually andcan cause recurrent genital ulcers (Fleming, D. T., McQuillan, G. M.,Johnson, R. E. et al., "Herpes simplex virus type 2 in the UnitedStates, 1976 to 1994", N. Eng. J. Med., (1997), 337:1105-1111; Arvin, A.M., Prober, C. G., "Herpes Simplex Virus Type 2--A Persistent Problem",N. Engl. J. Med., (1997), 337:1158-1159). The prevalence of HSV-2infections is even higher in some developing countries (Nahmias, A. J.,Lee, F. K., Beckman-Nahmias, S., "Sero-epidemiologial and SociologicalPatterns of Herpes Simplex Virus Infection in the World", Scand. J.Infect. Dis., (1990), Suppl. 69:19-36). Although the infection istreatable by antiviral drugs, efficacious long-term suppression ofgenital herpes is expensive (Engel, J. P., "Long-term Suppression ofGenital Herpes", JAMA, (1998), 280:928-929). The probability of furtherspread of the virus by untreated people and asymptomatic carriers notreceiving antiviral therapy is extremely high, considering the highprevalence of infections. Other herpesviruses, including cytomegalovirus(Krieger, J. N., Coombs, R. W., Collier, A. C. et al., "Seminal Sheddingof Human Immunodeficiency Virus Type 1 and Human Cytomegalovirus:Evidence For Different Immunologic Controls", J. Infect. Dis., (1995),171:1018-1022; van der Meer, J. T. M., Drew, W. L., Bowden R. A. et al.,"Summary of the International Consensus Symposium on Advances in theDiagnosis, Treatment and Prophylaxis of Cytomegalovirus Infection",Antiviral Res., (1996), 32:119-140) (HCMV), herpesvirus 6 (Leach, C. T.,Newton, E. R., McParlin, S. et al., "Human Herpesvirus 6 Infection ofthe Female Genital Tract", J. Infect. Dis., (1994), 169:1281-1283), andherpesvirus 8 (Howard, M. R., Whitby, D., Bahadur, G. et al., "Detectionof Human Herpesvirus 8 DNA in Semen from HIV-infected Individuals ButNot Healthy Semen Donors", AIDS, (1997), 11:F15-F19), the causativeagent of Kaposi's sarcoma, are also transmitted sexually.

The time of initial herpes simplex virus infection is usually obscureexcept in the uncommon primary systemic infection occurring in infantsand is characterized by generalized cutaneous and mucous membranelesions accompanied by severe constitutional symptoms. Localizedinfections ordinarily appear in childhood, but may be delayed untiladult life. It is presumed that the herpes simplex virus remains dormantin the skin and that herpetic eruptions are precipitated by overexposureto sunlight, febrile illnesses, or physical or emotional stress; also,certain foods and drugs have been implicated. In many instances, thetrigger mechanism remains undetected.

The lesions caused by herpes simplex virus may appear anywhere on theskin or on mucous membranes, but are most frequent on the face,especially around the mouth or on the lips, conjunctiva and cornea, orthe genitals. The appearance of small tense vesicles on an erythematousbase follows a short prodromal period of tingling discomfort or itching.Single clusters may vary from 0.5 to 1.5 cm in size, but several groupsmay coalesce. Herpes simplex on skin tensely attached to underlyingstructures (for example, the nose, ears or fingers) may be painful. Thevesicles may persist for a few days, then begin to dry, forming a thinyellowish crust. Healing usually occurs within 10 days after onset. Inmoist body areas, healing may be slower, with secondary inflammation.Healing of individual herpetic lesions is usually complete, butrecurrent lesions at the same site may result in atrophy and scarring.

In females infected with HSV-2, there may be no skin lesions, theinfection may remain entirely within the vagina. The cervix isfrequently involved, and there is increasing evidence that this may be afactor in the development of carcinoma of the cervix.

Corneal lesions commonly consist of a recurrent herpetic keratitis,manifest by an irregular dendritic ulcer on the superficial layers.Scarring and subsequent impairment of vision may follow.

Gingivostomatitis and vulvovaginitis may occur as a result of herpesinfection in infants or young children. Symptoms include irritability,anorexia, fever, inflammation, and whitish plaques and ulcers of themouth. Particularly in infants, though sometimes in older children,primary infections may cause extensive organ involvement and fatalviremia.

In women who have an attack of HSV-2 late in pregnancy, the infectionmay be transmitted to the fetus, with the development of severe viremia.Herpes simplex virus may also produce fatal encephalitis.

Kaposi's varicelliform eruption (eczema herpeticum) is a potentiallyfatal complication of infantile or adult atopic eczema. Exposure ofpatients with extensive atopic dermatitis to persons with active herpessimplex should be avoided.

No local or systemic chemotherapeutic agent has been demonstrated to beeffective for treating herpes simplex virus with the possible exceptionof topical idoxuridine (IDU) in superficial herpetic keratitis. Reportson this compound in cutaneous herpes are conflicting. Other drugs whichhave been employed to treat HSV include trifluorothymidine, vidarabine(adenine arabinoside, ara-A), acyclovir, and other inhibitors of viralDNA synthesis may be effective in herpetic keratitis. These drugsinhibit herpes simplex virus replication and may suppress clinicalmanifestations. However, the herpes simplex virus remains latent in thesensory ganglia, and the rate of relapse is similar in drug-treated anduntreated individuals. Moreover, some drug-resistant herpes virusstrains have emerged.

Diseases caused by varicella-zoster virus (human herpesvirus 3) includevaricella (chickenpox) and zoster (shingles).

Cytomegalovirus (human herpesvirus 5) is responsible for cytomegalicinclusion disease in infants. There is presently no specific treatmentfor treating patients infected with cytomegalovirus.

Epstein-Barr virus (human herpesvirus 4) is the causative agent ofinfectious mononucleosis and has been associated with Burkitt's lymphomaand nasopharyngeal carcinoma.

Animal herpesviruses which may pose a problem for humans include B virus(herpesvirus of Old World Monkeys) and Marmoset herpesvirus (herpesvirusof New World Monkeys).

In searching for inexpensive antiviral compounds which could be appliedtopically to decrease the frequency of sexual transmission of the humanimmunodeficiency virus type 1 (HIV-1) and herpesviruses (HSV),applicants decided against all odds to screen excipients for anti-HIV-1activity and discovered the present invention which involves theadministration of cellulose acetate phthalate ("CAP") or hydroxypropylmethylcellulose phthalate ("HPMCP").

d. Sexually Transmitted Diseases of Bacterial Origin

Curable sexually transmitted diseases (STDs) of bacterial origin are themost common worldwide cause of illness with significant health, socialand economic consequences. They can lead to long-term, seriouscomplications and sequelae. The estimated annual (1995) worldwideincidence of four major curable STDs, namely syphillis, gonorrhea(Neisseria gonorrhoeae), chlamydia and trichomoniasis, was about 330million (Gerbase, A. C., Rowley, J. T., Heymann, D. H. L. et al.,"Global Prevalence and Incidence Estimates of Selected Curable STDs",Sex. Transm. Inf., (1998), 74 (Suppl. 1): S12-S16). Another treatableSTD, chancroid, a genital ulcerative disease caused by Haemophilusducreyi, is common in developing countries in Africa, Asia and LatinAmerica, where incidence may exceed that of syphillis (Trees, D. L.,Morse, S. A., "Chancroid and Haemophilus ducreyi: An Update", Clin.Microb. Rev., (1995), 8:357-375). The proposed control measures forthese STDs include the following: surveillance, laboratory diagnosis,syndromic management, data monitoring, treatment with antibacterialagents, partner notification and development of vaccines (Rao, P.,Mohamedali, F. Y., Temmerman, M. et al., "Systematic Analysis of STDControl: an Operational Model", Sex. Transm. Inf., (1998), 74 (Suppl 1):S17-S22; Dallabetta, G. A., Gerbase, A. C., Holmes, K. K., "Problems,Solutions, and Challenges in Syndromic Management of SexuallyTransmitted Diseases", Sex. Transm. Inf., (1998), 74 (Suppl 1): S1-S11;Burstein, G. R., Gaydos, C. A., Diener-West M., "Incident ChlamydiaTrachomatis Infections Among Inner-city Adolescent Females", JAMA,(1998), 280:521-526).

There has thus heretofore been desired the development of a topicalmicrobicide from inexpensive, widely available resources, with broadantiviral and antibacterial activities.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a safe and relativelyinexpensive method to decrease the frequency of transmission of humanimmunodeficiency virus and herpesvirus viral infections, particularlythose which are sexually transmitted.

A further object of the present invention is to provide a compositionfor decreasing the frequency of transmission of human immunodeficiencyvirus and herpesvirus.

Another object of the present invention is to provide methods fortreating and preventing sexually transmitted bacterial infections.

The above objects, along with all objects, aims and disadvantages, areachieved by the present invention.

The present invention concerns a method for decreasing the frequency oftransmission and, particularly, preventing the transmission of humanimmunodeficiency virus or herpesvirus by administering to a human aneffective anti-human immunodeficiency virus or anti-herpesvirus amountof at least one cellulose phthalate selected from the group consistingof acetate phthalate (CAP) and hydroxypropyl methylcellulose phthalate(HPMCP) either alone, or in combination with a pharmaceuticallyacceptable carrier or diluent.

The present invention also concerns a pharmaceutical composition fordecreasing the frequency of transmission of human immunodeficiency virusor herpesvirus comprising an effective anti-immunodeficiency virusamount or effective anti-herpesvirus amount of at least one cellulosephthalate selected from the group consisting of cellulose acetatephthalate and hydroxypropyl methylcellulose phthalate in combinationwith a pharmaceutically acceptable carrier or diluent.

The present invention further relates to a method for preventing thetransmission of a sexually transmitted bacterial infection in a human,or treating a human infected with a sexually transmitted bacterialinfection comprising administering to the human an effectiveanti-bacterial amount of at least one cellulose phthalate selected fromthe group consisting of acetate phthalate (CAP) and hydroxypropylmethylcellulose phthalate (HPMCP) either alone, or in combination with apharmaceutically acceptable carrier or diluent.

The present invention also concerns a pharmaceutical composition forpreventing the transmission of or for treating a sexually transmittedbacterial infection comprising an effective anti-bacterial amount of atleast one cellulose phthalate selected from the group consisting ofcellulose acetate phthalate and hydroxypropyl methylcellulose phthalatein combination with a pharmaceutically acceptable carrier or diluent.

The present invention is also directed to the aforesaid methods andpharmaceutical compositions, wherein the cellulose phthalate (CAP and/orHPMCP) is provided in the form of a suspension and preferably in amicronized form. Further, such suspension may include a water miscible,essentially anhydrous, non-solvent for CAP or HPMCP, such as glycerol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of % inhibition vs. cellulose acetate phthalate("CAP") concentration, for HSV-1 and HSV-2. FIG. 1 thus shows theinhibitory effect of cellulose acetate phthalate ("CAP") on HSV-1 andHSV-2.

FIG. 2 is a graph of % inhibition vs. HPMCP concentration, for HSV-1 andHSV-2. The results shown in FIG. 2 are similar to those shown in FIG. 1.

FIG. 3 is a graph of HIV-1 p24 antigen (absorbance at 450 nm) vs. HIV-1dilution. FIG. 3 shows the disintegration of purified HIV-1 by treatmentwith an "AQUATERIC"-glycerol formulation ("CAP formulation I") with orwithout polyvinyl pyrrolidone (PVP) and Crospovidone for 5 minutes at37° C., as measured by the release of the nucleocapsid antigen p24.

FIG. 4 is a graph of HIV-1 p24 antigen (absorbance at 450 nm) vs. virusconcentration. FIG. 4 shows the inactivation of HIV-1 infectivity bytreatment with an "AQUATERIC"-glycerol formulation containing 286 mg/mlof "AQUATERIC" for 5 minutes at 37° C., as determined by production ofthe nucleocapsid antigen p24 by infected cells as measured by ELISA.

FIG. 5 is a graph of absorbance (410 nm) vs. virus dilution. FIG. 5shows the inactivation of HSV-1 and HSV-2 by a suspension of "AQUATERIC"in glycerol. Virus preparations were mixed 1:1 with a suspension of"AQUATERIC" in glycerol for 5 minutes at 37° C.

FIG. 6 is a graph of absorbance (410 nm) vs. virus dilution. FIG. 6shows the inactivation of HSV-1 and HSV-2 by an "AQUATERIC"-glycerolformulation with PVP and Crospovidone.

FIG. 7 is a graph which shows the disintegration of purified HIV-1 bytreatment with a formulation of micronized CAP in glycerol ("CAPformulation I", which is defined hereinbelow) for 5 minutes at 37° C. inthe presence of seminal fluid and whole blood, respectively (for furtherdetails see FIG. 3).

FIG. 8 is a graph which shows the inactivation of CMV by the CAPformulation I. Virus preparations were mixed 1:1 with CAP formulation Ifor 5 minutes at 37° C. Serial dilutions of the virus preparations weretested for infectivity using a readout system based on the quantitationof β-galactosidase (absorbance at 410 nm).

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the use of cellulose acetate phthalate(CAP) and/or hydroxypropyl methylcellulose phthalate (HPMCP) to preventthe transmission of viral infections and to prevent or treat sexuallytransmitted bacterial infections.

Some of the properties of CAP as described in the Handbook ofPharmaceutical Excipients are summarized as follows:

Non proprietary Names:

BP: Cellacephate

PhEur: Cellulosi acetas phthalas

USPNF: Cellulose acetate phthalate

Synonyms:

Acetyl phthalyl cellulose; CAP; cellacefate; cellulose acetate hydrogen1,2-benzenedicarboxylate; cellulose acetate hydrogen phthalate;cellulose acetate monophthalate; cellulose acetophthalate; celluloseacetylphthalate.

Chemical Name and CAS Registry Number:

Cellulose, acetate, 1,2-benzenedicarboxylate [9004-38-0] Celluloseacetate phthalate is a cellulose in which about half the hydroxyl groupsare acetylated and about a quarter are esterified, with one of the twoacid groups being phthalic acid. The other acid group is free. See thestructural formula below. ##STR1## Functional Category: Coating agent.

Applications in Pharmaceutical Formulation or Technology:

Cellulose acetate phthalate has heretofore been used as an enteric filmcoating material, or as a matrix binder, for tablets and capsules(Spitael, J., Kinget, R., Naessens, K., "Dissolution Rate of CelluloseAcetate Phthalate and Bronsted Catalysis Law", Pharm. Ind., (1980),42:846-849; Takenaka, H., Kawashima, Y., Lin, S-Y., "Preparation ofEnteric-Coated Microcapsules for Tableting by Spray-Drying Technique andin vitro Simulation of Drug Release from the Tablet in GI Tract", J.Pharm., Sci., (1980), 69:1388-1392; Stricker, H., Kulke, H., "Rate ofDisintegration and Passage of Enteric-Coated Tablets in GastrointestinalTract", Pharm. Ind., (1981), 43:1018-1021; Takenaka, H., Kawashima, Y.,Lin, S-Y, "Polymorphism of Spray-Dried MicroencapsulatedSulfamethoxazole with Cellulose Acetate Phthalate and Colloidal SilicaMontmorillonite, or Talc", J. Pharm. Sci., (1981), 70:1256-1260;Maharaj, I., Nairn, J. G., Campbell J. B., "Simple Rapid method for thePreparation of Enteric-Coated Microspheres", J. Pharm. Sci., (1984),73:39-42; Beyger, J. W., Nairn, J. G., "Some Factors Affecting theMicroencapsulation of Pharmaceuticals with Cellulose Acetate Phthalate",J. Pharm. Sci., (1986), 75-573-578; Lin, S-Y, Kawashima, Y., "DrugRelease from Tablets Containing Cellulose Acetate Phthalate as anAdditive or Enteric-Coating Material", Pharm. Res., (1987), 4:70-74;Thoma, K. Hekenmuller, H., "Effect of Film Formers and Plasticizers onStability of Resistance and Disintegration Behaviour, Part 4:Pharmaceutical-Technological and Analytical Studies of Gastric JuiceResistant Commercial Preparations", Pharmazie, (1987), 42:837-841).

Such coatings resist prolonged contact with the strongly acidic gastricfluid, but soften and swell in the mildly acidic or neutral intestinalenvironment.

Cellulose acetate phthalate, when heretofore used as an adjuvant, wascommonly applied to solid dosage forms either by coating from organic oraqueous solvent systems, or by direct compression. Concentrations usedwere 0.5 to 9.0% of the core weight. The addition of plasticizersimproves the water resistance of this coating material, and suchplasticized films are more effective than when cellulose acetatephthalate is used alone as an adjuvant. Cellulose acetate phthalate iscompatible with the following plasticizers: acetylated monoglyceride;butyl phthalylbutyl glycolate; dibutyl tartrate; diethyl phthalate;dimethyl phthalate; ethyl phthalyethyl glycolate; glycerin; propyleneglycol; triacetin; triacetin citrate and tripropionin. Cellulose acetatephthalate has also been used heretofore in combination with othercoating agents to control drug release, e.g., ethylcellulose.

Description:

Cellulose acetate phthalate is a hygroscopic, white, free-flowing powderor colorless flakes. It is tasteless and odorless, or may have a slightodor of acetic acid.

Pharmacopeial Specifications:

    ______________________________________                                                                    USPNF XVII                                        Test            PhEur 1984  (Suppl 2)                                         ______________________________________                                        Identification  +           +                                                 Appearance of solution                                                                        +           -                                                 Appearance of a film                                                                          +           -                                                 Solubility of a film                                                                          +           -                                                 Viscosity at 25° C.                                                                    -           45-90 cP                                          Water           ≦5.0%                                                                              ≦5.0%                                      Residue on ignition                                                                           -           ≦0.1%                                      Sulfated ash    ≦0.1%                                                                              -                                                 Free acid       ≦3.0%                                                                              ≦6.0%                                      Heavy metals    ≦10 ppm                                                                            -                                                 Phthalyl content                                                                              30.0-40.0%  30.0-36.0%                                        Acetyl content  17.0-26.0%  21.5-26.0%                                        ______________________________________                                    

Typical Properties:

Hygroscopicity: cellulose acetate phthalate is hygroscopic andprecautions are necessary to avoid excessive absorption of moisture(Callahan, J. C., Cleary, G. W., Elefant, M., Kaplan, G., Kensler, T.,Nash, R. A., "Equilibrium Moisture Content of PharmaceuticalExcipients", Drug Dev. Ind. Pharm., (1982), 8:355-369).

Melting point: 192° C. Glass transition temperature is 160-170° C.(Sakellariou, P., Rowe, R. C., White, E. F. T., "The ThermomechanicalProperties and Glass Transition Temperatures of Some CelluloseDerivatives used in Film Coating", Int. J. Pharmaceutics, (1985),27:267-277).

Solubility: practically insoluble in alcohols, chlorinated hydrocarbons,hydrocarbons, and water; soluble in cyclic ethers, esters, etheralcohols, ketones and certain solvent mixtures. Also soluble in certainbuffered aqueous solutions at greater than pH 6. The following listshows some of the solvents and solvent mixtures in which celluloseacetate phthalate has a solubility of 1 in 10 parts or more.

Acetone

Acetone: Ethanol (1:1)

Acetone: Methanol (1:1/1:3)

Acetone: Methylene chloride (1:1/1:3)

Acetone: Water (97:3)

Benzene: Methanol (1:1)

Diacetone alcohol

Dioxane

Ethoxyethyl acetate

Ethyl acetate: Ethanol (1:1)

Ethyl acetate: Propan-2-ol (1:1/1:3)

Ethylene glycol monoacetate

Ethyl lactate

Methoxyethyl acetate

β-Methoxyethylene alcohol

Methyl acetate

Methylene chloride: Ethanol (3:1)

Methyl ethyl ketone

Viscosity (dynamic): 50-90 mPas (50-90 cP) for a 15% w/w solution inacetone with a moisture content of 0.4%. This is a good coating solutionwith a honey-like consistency, but the viscosity is influenced by thepurity of the solvent.

Stability and Storage Conditions:

Cellulose acetate phthalate hydrolyzes slowly under prolonged adverseconditions, such as high temperature and humidity, with a resultantincrease in free acid content, viscosity and odor of acetic acid. If itsmoisture content is above about 6% w/w, fairly rapid hydrolysis occurs.However, cellulose acetate phthalate is stable if stored in awell-closed container in a cool, dry place.

Incompatibilities:

Cellulose acetate phthalate is incompatible with ferrous sulfate, ferricchloride, silver nitrate, sodium citrate, aluminum sulfate calciumchloride, mercuric chloride, barium nitrate, basic lead acetate, andstrong oxidizing agents such as strong alkalis and acids. It should benoted that one carboxylic acid group of the phthalic acid moiety remainsunesterified and free for interactions. Accordingly, incompatibilitywith acid sensitive drugs may occur (Rawlins E. A., editor, "Bentley'sTextbook of Pharmaceutics", London: Bailliere, Tindall and Cox, (1977),291).

Method of Manufacture:

Cellulose acetate phthalate is produced by reacting the partial acetateester of cellulose with phthalic anhydride in the presence of a tertiaryorganic base, such as pyridine.

Safety:

Cellulose acetate phthalate is widely used in oral pharmaceuticalproducts and is generally regarded as a nontoxic material, free ofadverse effects.

Results of long-term feeding studies with cellulose acetate phthalate,in rats and dogs, have indicated a low oral toxicity. Rats surviveddaily feedings of up to 30% in the diet for up to one year withoutshowing a depression in growth. Dogs fed 16 g daily in the diet for oneyear also remained normal (Hodge, H. C., "The Chronic Toxicity ofCellulose Acetate Phthate in Rats and Dogs", J. Pharmacol., 80, 250-255,(1944)).

Regulatory Status:

Included in the FDA Inactive Ingredients Guide (oral capsules andtablets). Included in nonparenteral medicines licensed in the UnitedKingdom.

Pharmacopeias: Aust, Br, Braz, Cz, Eur, Fr, Ger, Gr, Hung, Ind, It, Jpn,Mex, Neth, Nord, Port, Swiss and USPNF.

Some of the properties of HPMCP, described in the Handbook ofPharmaceutical Excipients are summarized as follows:

Non proprietary Names: BP: Hypromellose phthalate; PhEur:Methylhydroxypropylcellulosi phthalas and USPNF: Hydroxypropylmethylcellulose phthalate.

Synonyms: Cellulose phthalate hydroxypropyl methyl ether; HPMCP;2-hydroxypropyl methylcellulose phthalate; methylhydroxypropylcellulosephthalate.

Chemical Name and CAS Registry Number: Cellulose, hydrogen1,2-benzenedicarboxylate, 2-hydroxypropyl methyl ether [9050-31-1]

Structural Formula: ##STR2## Functional Category: Coating agent.

Applications in Pharmaceutical Formulations or Technology

Hydroxypropyl methylcellulose phthalate has heretofore been widely usedin oral pharmaceutical formulations as an enteric coating material fortablets or granules (Ehrhardt, L., Patt, L., Schindler, E.,"Optimization of Film Coating Systems", Pharm. Ind., (1973), 35:719-722;Delporte, J. P., Jaminet, F., "Influence of Formulation ofEnteric-Coated Tablets on the Bioavailability of the Drug", J. Pharm.Belg., (1976), 31-263-276; Patt, L., Hartmann V., "Solvent Residues inFilm Forming Agents", Pharm. Ind., (1976), 38:902-906; Stafford, J. W.,"Enteric Film Coating Using Completely Aqueous Dissolved HydroxypropylMethylcellulose Phthalate Spray Solutions", Drug. Dey Ind. Pharm.,(1982), 8:513-530; Thoma, K., Heckenmuller, H., Oschmann, R.,"Resistance and Disintegration Behaviour of Gastric Juice ResistantDrugs", Pharmazie, (1987), 42:832-836; Thoma, K., Heckenmuller, H.,Oschmann, R., "Impact of Film Formers and Plasticizers on Stability ofResistance and Disintegration Behaviour", Pharmazie, (1987),42:837-841).

Hydroxypropyl methylcellulose phthalate is insoluble in gastric fluid,but will swell and dissolve rapidly in the upper intestine. Generally,concentrations of 5-10% of hydroxypropyl methylcellulose phthalate wereemployed with the material being dissolved in either adichloromethane:ethanol (50:50) or an ethanol:water (80:20) solventmixture. Hydroxypropyl methylcellulose phthalate can normally be appliedto tablets and granules without the addition of a plasticizer or otherfilm formers, using established coating techniques (Rowe, R. C.,"Molecular Weight Studies on the Hydroxypropyl Methylcellulose Phthalate(HP55)", Acta. Pharm. Technol., (1982), 28(2):127-130. However, theaddition of a small amount of plasticizer or water can avoid filmcracking problems; many commonly used plasticizers such as diacetin,triacetin, diethyl and dibutyl phthalate, castor oil, acetylmonoglyceride and polyethylene glycols are compatible with hydroxypropylmethylcellulose phthalate. Tablets coated with hydroxypropylmethylcellulose phthalate disintegrate more rapidly than tablets coatedwith cellulose acetate phthalate.

Hydroxypropyl methylcellulose phthalate can be applied to tabletsurfaces using a dispersion of the micronized hydroxypropylmethylcellulose phthalate powder in an aqueous dispersion of a suitableplasticizer such as triacetin, triethyl citrate or diethyl tartratealong with a wetting agent (Muhammad, N. A., Boisvert, W., Harris, M.R., Weiss, J., "Evaluation of Hydroxypropyl Methylcellulose Phthalate 50as Film Forming Polymer from Aqueous Dispersion Systems", Drug Dev. Ind.Pharm., (1992), 18:1787-1797).

Hydroxypropyl methylcellulose phthalate may be used alone or incombination with other soluble or insoluble binders in the preparationof granules with sustained drug release properties; the release rate ispH dependent. Since hydroxypropyl methylcellulose phthalate is tastelessand insoluble in saliva, it can be used as a coating to mask theunpleasant taste of some tablet formulations.

Description:

Hydroxypropyl methylcellulose phthalate occurs as white to slightlyoff-white colored free-flowing flakes or as a granular powder. It isodorless or with a slightly acidic odor, and a barely detectable taste.

Typical Properties:

Melting point: 150° C.

Solubility: practically insoluble in ethanol and water; very slightlysoluble in acetone, and toluene; soluble in aqueous alkalis, a mixtureof equal volumes of acetone and methanol, and in a mixture of equalvolumes of dichloromethane and methanol.

Stability and Storage Conditions:

Hydroxypropyl methylcellulose phthalate is chemically and physicallystable at ambient temperature and humidity for 3-4 years, and for 2 to 3months at 40° C. and 75% relative humidity (Shin-Etsu Chemical Co.,Ltd., Technical Literature: Hydroxypropyl Methylcelluose Phthalate,(1993). Hydroxypropyl methylcellulose phthalate is stable on exposure toUV light for up to 3 months at 25° C. and 70% relative humidity(Shin-Etsu Chemical Co., Ltd., Technical Literature: HydroxypropylMethylcelluose Phthalate, (1993). In general, hydroxypropylmethylcellulose phthalate is more stable than cellulose acetatephthalate. At ambient storage conditions, hydroxypropyl methylcellulosephthalate is not susceptible to microbial attack.

Incompatibilities:

Incompatible with strong oxidizing agents. Splitting of film coatingshas been reported rarely, most notably with coated tablets which containmicrocrystalline cellulose and calcium carboxymethylcellulose. Filmsplitting has also occurred when a mixture of acetone: propan-2-ol ordichloromethane: propan-2-ol has been used as a coating solvent, or whencoatings have been applied in conditions of low temperature andhumidity. However, film splitting may be avoided by careful selection ofthe coating solvent used, by using a higher molecular weight grade ofpolymer (Rowe, R. C., "Molecular Weight Studies on the HydroxypropylMethylcellulose Phthalate (HP55), Acta. Pharm. Technol., (1982),28(2):127-130), or by the addition of a plasticizer, such as acetylmonoglyceride or triacetin. The addition of more than about 10% titaniumdioxide to a coating solution of hydroxypropyl methylcellulosephthalate, that is used to produce a colored film coating, may result incoatings with decreased elasticity and gastric fluid resistance(Shin-Etsu Chemical Co., Ltd., Technical Literature: HydroxypropylMethylcellulose Phthalate, (1993)).

Method of Manufacture:

Hydroxypropyl methylcellulose acetate phthalate is prepared by theesterification of hydroxypropyl methylcellulose with phthalic anhydride.The degree of methoxy and phthalyl substitution determines theproperties of the polymer and in particular the pH at which it dissolvesin aqueous media.

Safety:

Hydroxypropyl methylcellulose phthalate has been heretofore widely used,primarily as an enteric coating agent, in oral pharmaceuticalformulations. Chronic and acute animal feeding studies on severaldifferent species have shown no evidence or teratogenicity or toxicityassociated with hydroxypropyl methylcellulose phthalate (Kitagawa, H.,Kawana, H., Satoh, T., Fukuda, Y., "Acute and Subacute Toxicities ofHydroxypropyl Methylcellulose Phthalate", Pharmacometrics, (1970),4(6):1017-1025; Kitagawa, H., Satoh, T., Yokoshima, T., Nanbo, T.,"Absorption, Distribution and Excretion of Hydroxypropyl MethylcellulosePhthalate in the Rat", Pharmacometrics, (1971), 5(1):1-4; Ito, R.,Toida, S., "Studies on the Teratogenicity of a New Enteric CoatingMaterial, Hydroxypropyl Methylcellulose Phthalate (HPMCP) in Rats andMice", J. Med. Soc. Toho-Univ., (1972), 19(5):453-461; Kitagawa, H.,Yano, H., Fukuda, Y., "Chronic Toxicity of HydroxypropylmethylcellulosePhthalate in Rats", Pharmacometrics, (1973), 7(5);689-701; Kitagawa, H.,Yokoshima, T., Nanbo, T., Hasegawa, M., "Absorption, Distribution,Excretion and Metabolism of ¹⁴ C-hydroxypropyl MethylcellulosePhthalate", Pharmacometrics, (1974), 8(8):1123-1132. Hydroxypropylmethylcellulose phthalate is generally regarded as a nonirritant andnontoxic material.

LD₅₀ (rat, oral): >15 g/kg (Kitagawa et al., Pharmacometrics, (1970),4(6):1017-1025).

Regulatory Status: included in the FDA Inactive Ingredients Guide (oralcapsules and tablets) and included in nonparenteral medicines licensedin the United Kingdom.

Pharmacopeias: Br, Eur, Fr, Gr, It, Jpn, Neth, Port, Swiss and USPNF.

Related Substances: cellulose acetate phthalate; HydroxypropylMethylcellulose.

A particularly preferred composition for topically administering to ahuman in accordance with the present invention comprises a micronizedpreparation containing CAP or micronized HPMCP, a poloxamer anddistilled acetylated monoglycerides (a mixture of micronized CAP,poloxamer and acetylated monoglycerides is sold by the FMC Corporationunder the trade name "AQUATERIC") suspended in glycerol. A poloxamer isa nonionic polyoxyethylene-polyoxypropylene copolymer. Squalane(2,6,10,15,19,23-hexamethyltetracosane) can be used instead of glycerol.

A chemical name for a poloxamer is α-hydro-ω-hydroxypoly(oxyethylene)poly(oxypropylene) poly(oxyethylene) block copolymer. The poloxamerpolyols are a series of closely related block copolymers of ethyleneoxide and propylene oxide conforming to the following formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H.

    ______________________________________                                        The following is a list of grades of poloxamers (USPNF XVII):                           Physical             Average Molecular                              Poloxamer Form    a        b   Weight                                         ______________________________________                                        124       Liquid  12       20  2,090 to 2,360                                 188       Solid   80       27  7,680 to 9,510                                 237       Solid   64       37  6,840 to 8,830                                 338       Solid   141      44  12,700 to 17,400                               407       Solid   101      56   9,840 to 14,600                               ______________________________________                                    

To prevent separation from the glycerol of the microsuspensioncontaining the CAP or HPMCP, the poloxamer and the distilled acetylatedmonoglycerides, it is preferred to add polyvinylpyrrolidone ("PVP") anda 1-ethenyl-2-pyrrolidinone homopolymer (Crospovidone) (Polyplasdone)(C₆ H₉ NO)_(n), molecular weight>1,000,000) (water insoluble syntheticcross-linked homopolymer of N-vinyl-2-pyrrolidinone).

The term micronized used herein refers to particles having a particlesize of less than 35 microns, preferably less than 15 microns, morepreferably less than 10 microns and most preferably less than 5 microns.

In the composition described herein which includes glycerol, theglycerol may be replaced with a saline solution or water, so long as thecomposition is stored at ≦25° C.

CAP is commonly used as an enteric film coating material or as a matrixbinder for tablets and capsules. Its safety has been extensively studiedand it has been shown to be free of adverse effects. Vaginal irritationtests in the rabbit model further confirmed its safety. CAP is a highmolecular weight compound (M_(w) is approximately 60,000), indicatingthat if topically applied, it will not spread systemically. Thelikelihood of CAP spreading beyond the site of application has beenfurther decreased by using it in a micronized form.

Formulations of CAP were discovered by the applicants to be activeagainst HIV-1, herpesviruses, HSV-1, and HSV-2, cytomegalovirus,Chlamydia trochomatis, garderella, Neisseria gonorrhoeae, Haemophilusducreyi, and Trichomonas vaginalis.

On the other hand, the CAP formulations did not affect the viability ofLactobacilli which are essential components of the natural vaginal floraimortant for resistance against some STD pathogens (Hawes, S. E.,Hillier, S. L., Benedetti, J. et al., "Hydrogen Peroxide-ProducingLactobacilli and Acquisition of Vaginal Infections", J. Infect. Dis.,(1995), 172, 756-763).

Results indicated that the formulation did not affect the infectivity ofpapillomaviruses, another STD pathogen, involved in cervical cancer(Franco, E. L, Villa, L. L., Ruiz, A. et al., "Transmission of CervicalHuman Papillomavirus Infection by Sexual Activity: Differences BetweenLow and High Oncogenic Risk Types", J. Infect. Dis., (1995), 172,756-763; Bosch, F. X., Munoz, N., de Sanjose, S. et al., "Importance ofHuman Papillomavirus Endemicity in the Incidence of Cervical Cancer: AnExtension of the Hypothesis on Sexual Behavior", Cancer Epidemiol.Biomarkers & Prev., (1994), 3, 375-379).

Without wishing to be bound by any particular theory of operability, itis considered that the anti-viral and anti-bacterial activity of CAD maybe atributable to the hydrophobic nature of the phthalate residues onthe CAP polymer for its activity appears to be important againstpathogens of sexually transmitted diseases, since many other cellulosederivatives tested lacked both anti-HIV-1 and anti-herpesvirus activity.

A preferred composition for administration in the present invention canbe made as follows: dissolve PVP in glycerol, then add cross-linked1-ethenyl-2-pyrrolidinone homopolymer (Crospovidone) (Crospovidone iscross-linked povidone) and a composition comprising micronized CAP andpoloxamer and acetylated monoglycerides. The PVP and cross-linked1-ethenyl-2-pyrrolidinone homopolymer would be in concentrationssufficient to stabilize the suspension of "AQUATERIC" in glycerol.Squalane can be used instead of glycerol.

The method of the present invention can be used to prevent thetransmission of human immunodeficiency virus, such as HIV-1 and HIV-2,and herpesvirus, in humans. The present invention is thus effective forpreventing the transmission of HIV-1, or HSV, such as HSV-1, HSV-2,HSV-7 and HSV-8, as well as human cytomegalovirus, varicella-zostervirus, Epstein-Barr virus and human herpesvirus 6. Preferred embodimentsof the present invention are for preventing the transmission of HIV-1,HSV-1, or HSV-2, which are known to be transmitted sexually and HSV-8,which is known to be a causative agent of Kaposi's sarcoma.

The present invention also concerns preventing the transmission of ortreating a sexually transmitted bacterial infection such as syphillis,gonorrhea, chlamydia, trichomoniasis or an infection caused bygarderella vaginalis.

In the methods of the present invention for preventing the transmissionof HIV or herpesvirus infection in a human or for preventing thetransmission of or for treating a sexually transmitted bacterialinfection in a human, a pharmaceutically effective anti-viral amount oranti-bacterial amount, respectively, of CAP or HPMPC or both CAP andHPMPC is administered to a human. The composition for use in the presentinvention is administered to an appropriate region of the human body.

The phrase "administration to an appropriate region of the body"includes, for example, application of the (active ingredient (CAP orHPMPC or both) or a composition containing the same used to regions ofthe body of a human, for example, the region of the human body whichcomes into close contact with another human body, for example,application (directly or indirectly) to the male or female genitalia toprevent transmission of HIV-1, HSV-1, HSV-2 or bacterial infectionduring sexual intercourse.

The term "local administration" includes any method of administration inwhich the activity of the CAP or HPMCP or both used in the presentinvention is substantially confined to the region of the human's body towhich it is applied, i.e., vaginal or rectal (topical) administration.

The present invention is thus particularly effective for providing amethod of preventing the transmission of a viral infection such as HIVor herpesvirus infection or preventing the transmission of or treating abacterial infection which is transmitted by sexual contact, such asvaginal transmission, either during sexual intercourse or duringchildbirth (vaginal delivery), by vaginal administration, such as byadministering a cream, ointment, lotion, jelly, solution, emulsion orfoam formulation containing a pharmaceutically effective anti-HIV-1amount or anti-HSV amount or anti-bacterial amount of CAD (such asmicronized CAP) or HPMCP (such as micronized HPMCP) or both, eitheralone or in combination with a pharmaceutically acceptable carrier ordiluent.

To prevent transmission of HIV-1 or herpesvirus infection or a bacterialinfection which is transmitted by sexual contact, CAP or HPMCP (inmicronized form) or both can be applied to a contraceptive device (forexample, a male or female condom, a contraceptive diaphragm or acontraceptive sponge, for example, a polyurethane foam sponge), prior tosexual intercourse.

Alternatively, CAP or HPMCP or both can be applied on a pessary ortampon for vaginal administration. The pharmaceutical formulation fortopical administration would comprise a pharmaceutically effectiveanti-HIV amount or anti-herpesvirus amount or anti-bacterial amount ofCAP or HPMCP or both and at least one pharmaceutically acceptabletopical carrier or diluent, to form an ointment, cream, gel, lotion,paste, jelly, spray or foam.

The amount (dosage) of the active ingredient (CAP or HPMCP or both) in atopical formulation for use in the present invention will, in general,be less than 1,000 milligrams, preferably between 200 to 800 milligrams.

It is preferable to administer the active ingredient in conjunction witha pharmaceutically acceptable diluent or carrier, as a pharmaceuticalformulation. The present invention thus also involves the use of apharmaceutical formulation or composition comprising the activeingredient together with one or more pharmaceutically acceptablecarriers or diluents and, optionally, other prophylactic ingredients.The carrier(s) or diluent(s) should be "acceptable" in the sense ofbeing compatible with the other ingredients of the formulation and notdeleterious to the recipient.

Pharmaceutical formulations include those suitable for vaginal, rectalor topical administration. The formulations may, where appropriate, beconveniently presented in discrete dosage units and may be prepared byany of the methods well known in the art of pharmacy. All such methodsinclude the step of bringing into association the active ingredient withliquid carriers, gels or finely divided solid carriers or both and then,if necessary, shaping the product into the desired formulation.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, jelly, foams or sprays oraqueous or oily suspensions, solutions or emulsions (liquidformulations) containing in addition to the active ingredient, suchcarriers as are known in the art to be appropriate. These formulationsare useful to protect not only against sexual transmission of HIV or HSVor a sexually transmitted bacterial infection, but also to preventinfection of a baby during passage through the birth canal. Thus thevaginal administration can take place prior to sexual intercourse,during sexual intercourse, and immediately prior to childbirth.

As a vaginal formulation, the active ingredient may be used inconjunction with a spermicide and as discussed above, may be employedwith a condom, a diaphragm, a sponge or other contraceptive device.

Pharmaceutical formulations and preparations suitable for administrationmay conveniently be presented as a solution, an aqueous or oilysuspension, or an emulsion. The active ingredient may also be presentedas a bolus, electuary or paste.

Liquid preparations for vaginal administration may contain conventionaladditives such as suspending agents, emulsifying agents, non-aqueousvehicles (which may include edible oils) or preservatives.

Pharmaceutical formulations suitable for rectal administration, whereinthe carrier is a solid, are most preferably represented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping in molds.

Drops may be formulated with an aqueous or non-aqueous base comprisingone or more dispersing agents, solubilizing agents or suspending agents.Liquid sprays are conveniently delivered from pressurized packs.

When desired, the above described formulations adapted to give sustainedrelease of the active ingredient may be employed.

The pharmaceutical compositions for use according to the invention mayalso contain other active ingredients such as spermicides, orantimicrobial agents, preservatives or other anti-viral agents.

The micronized CAP and its suspension in glycerol or squalane accordingto the present invention results in an active and stable formulationwhich has antiviral activity and is suitable for topical application toprevent the sexual transmission of HIV-1, herpesviruses and sexuallytransmitted bacterial infections.

The present invention also relates to pharmaceutical compositionscontaining 1 to 25 wt. %, preferably 5 to 20 wt. % and, more preferably,12 to 18 wt. % of micronized cellulose acetate phthalate and glyceroland either polyvinylpyrrolidone and Crospovidone(1-ethenyl-2-pyrrolidinone homopolymer), or colloidal silicon dioxide,to form an easily applicable homogeneous cream.

In addition to the two formulations disclosed above, a semi-solidformulation containing all solid ingredients (i.e., "AQUATERIC",povidone, Crospovidone and colloidal silicone dioxide) can be mixed withglycerol or squalane wherein the amounts of all the comoponents aresufficient to result in a semi-solid dough or putty which can be easilyshaped, aliquotted into desired portions and packaged for protectionfrom environmental factors (humidity, etc.).

Moreover, the "AQUATERIC" can be replaced by another form of micronizedCAP. This could be obtained, for example, by dissolving 100 mg of CAPand 100 mg of polyvinylpyrrolidone (povidone, PVP) per 1 ml of dimethylsulfoxide. After dissolution of the solid components, water is slowlyadded under efficient, vigorous mixing. This will result in theformation of a fine precipitate of CAP containing PVP. The precipitateis subsequently washed with water and finally freeze-dried. The fine,freeze-dried powder can be used instead of "AQUATERIC".

The procedure disclosed in the preceding paragraph is much simpler thana similar procedure utilizing polyvinyl alcohol instead of PVP, andacetone instead of dimethyl sulfoxide, and also requires the presence ofa mineral salt, such as magnesium chloride (U.S. Pat. No. 4,968,350 toBindschaedler et al., "Process for Preparing a Powder of Water-insolublePolymer which can be Redispersed in a Liquid Phase, the Resulting Powderand Utilization Thereof"). The presence of Mg⁺⁺ is also undesirable,since it decreases the stability of CAP.

The aforementioned formulation of "AQUATERIC", PVP and Crospovidone inglycerol is suitable for topical application. However, in order to apply(administer) the formulation in predetermined quantities, in addition tothe formulation, a measuring device, e.g., an applicator, should beprovided.

It will be advantageous to incorporate the formulations containing CAPand/or HPMCP into hydroxypropyl methylcellulose capsules such as "VEGICAPS" or "VEGGIE-CAPS", manufactured by GS Technologies, Springville,Utah, which can be configured as vaginal suppositories. This wouldreduce costs and avoid possible disposal problems. Such suppositoriescan be inserted into the vagina intact, whereby the shell of the capsulewill soften and rupture upon interaction with moisture within thevagina, thus releasing the CAP and/or HPMCP formulation.

For example, the above-described hydroxypropyl methylcellulose capsulescan be filled either with:

(a) "AQUATERIC" suspended in glycerol; or

(b) "AQUATERIC" in solid form, with or without additional inactiveingredients, can also be incorporated in gelatin capsules.

The formulation containing the active ingredient (CAD and/or HPMCP) ofthe present invention can be in the form of a single capsule or theformulation may be in the form of two or more capsules, each containingthe same or distinct ingredients.

Applicants discovered that two of many pharmaceutical excipients displaya potent anti-HIV-1 activity effect and anti-bacterial effect. This isof enormous importance since excipients are inexpensive compounds. Theexpected dose of CAP or HPMCP per single topical application(approximately 300 mg) is expected to cost approximately 1.33 US cents.Thus, the application of CAP and/or HPMCP for decreasing the frequencyof sexual transmission of HIV-1 and bacterial infections is economicallyfeasible worldwide and is expected to contribute to the control of theworldwide HIV-1 epidemic.

Since viruses other than HIV-1 are also transmitted sexually, it was ofinterest to determine whether CAD and/or HPMCP may also inhibitinfection by such viruses. Herpesvirus type 1 (HSV-1) and type 2 (HSV-2)were selected for these experiments. Results summarized in FIG. 1indicate that CAP inhibited infection by both HSV-1 and HSV-2. Similarresults were obtained with HPMCP (see FIG. 2).

Some of the results presented herein were briefly summarized under thecode "microbicide B195" at the 12th World AIDS Conference, Neurath, A.R., Strick, N., Lin, K. et al., "Microbicide B195", Proceedings of the12th World AIDS Conference, Geneva, Switzerland, Jun. 28-Jul. 3, 1998,pp. 239-242.

EXAMPLES Example 1:

Screening Pharmaceutical Excipients for Anti-HIV Activity

All compounds were first screened for anti-HIV-1 activity by measuringthe inhibition of fusion between HIV-1 infected and uninfected cells(Jiang, S., Lin, K., Strick, N., Neurath, A. R., "Inhibition of HIV-1Infection by a Fusion Domain Binding Peptide from the HIV-1 EnvelopeGlycoprotein gp41", Biochem. Biophys. Res. Commun. (1993), 195,533-538).

The selection of pharmaceutical excipients to screen for anti-HIVactivity was made from a list of pharmaceutical excipients derived fromthe Handbook of Pharmaceutical Excipients, edited by Ainley Wade andPaul J. Weller, 2^(nd) edition, American Pharmaceutical Association,Washington, D.C., and the Pharmaceutical Press, London, (1994). Theselected compounds are listed in the following Table 1A. Otherexcipients listed in the Handbook of Pharmaceutical Excipients were nottested for anti-HIV-1 activity, since it was known from earlier studiesthat they do not have such activity (see the following Table 2).Compounds insoluble in water or buffers (see the following Table 3),organic compounds including oils, waxes, solvents and detergents knownto solubilize cell membranes and envelopes of lipid-containing viruses(see the following Table 4), gases used for aerosol propellants (see thefollowing Table 5), and oxidizing agents and disinfectants withantibacterial activity (see the following Table 6) were excluded fromthe screening process.

Surprisingly, of all the compounds listed in Table 1A, only twocompounds inhibited fusion between HIV-1 infected and uninfected cells,corresponding to a method for rapidly assessing the anti-HIV-1 activityof compounds. In this assay, HIV-1 IIIB infected H9 cells were labeledby 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester (BCECF; Molecular Probes, Inc., Eugene, Oreg.) according to themanufacturer's instructions. BCECF-labeled H9/HIV-1 IIIB cells (10⁴)were mixed with 2×10⁵ uninfected MT-2 cells. After incubation in a96-well plate at 37° C. for 1 hour, the fused and unfused labeled cellswere counted under an inverted fluorescence microscope at a160×magnification. At least 200 BCECF-labeled cells were counted and theproportion of fused cells was determined. These tests were carried outin the presence and absence of graded quantities of compounds to bescreened. All experiments with HIV-1 were carried out under P2 biohazardcontainment levels.

The anti-HIV-1 activity of the two compounds, namely cellulose acetatephthalate and hydroxypropyl methylcellulose phthalate, listed in Table1A was confirmed and quantitated by the following additional tests: (a)inhibition of the cytopathic effect (CPE) of HIV-1 and (b) inhibition ofproduction of the HIV-1 nucleocapsid antigen (p24) (Neurath, A. R.,Strick, N., Haberfield, P., Jiang, S., "Rapid Prescreening for AntiviralAgents Against HIV-1 Based on their Inhibitory Activity in Site-DirectedImmunoassays, II. Porphyrins Reacting with the V3 loop of gp120",Antivir. Chem. Chemother., (1992), 3, 55-63) (Table 1B). The twocompounds were not toxic for uninfected cells at concentrations ≦2,500μg/ml.

10⁴ MT-2 cells in 96-well plates were infected with HIV-1 (a dosesufficient to accomplish a multiplicity of injection of 0.0045) in 200μl of RPMI 1640 medium supplemented with 10 vol. % fetal bovine serum("FBS"). After 1 hour and 24 hours, half of the culture medium waschanged and replaced by fresh medium. On the fourth day after incubationat 37° C., 100 μl of culture supernatants were collected from each welland an equal volume of fresh medium was added to the wells. Thecollected supernatants were mixed with an equal volume of 5 vol. %TRITON X-100 and assayed for the p24 antigen using an ELISA(Enzyme-linked immunoassay) kit from Coulter Immunology (Hialeah, Fla.).On the sixth day after infection, an indicator, XTT Tetrazolium Dye (1mg/ml; 50 μl/well; PolySciences, Inc., Warrington, Pa.) was added to thecells. After 4 hours, intracellular formazan was determinedcolorometrically at 450 nm following the described procedure (Weislow O.S., Kiser, R., Fine, D. L. et al., "New Soluble-Formazan Assay for HIV-1Cytopathic Effects: Application to High-Flux Screening of Synthetic andNatural Products for AIDS-Antiviral Activity", J. Natl. Cancer Inst.,81, 577-586, (1989)). The percentage of cytopathogenesis was calculatedusing the following formula: 100×[(OD₄₅₀ in negative control-OD₄₅₀ inexperiment)/(OD₄₅₀ in negative control-OD₄₅₀ in positive control)]. Thenegative control corresponded to cells mixed with culture medium,instead of HIV-1, while the positive control represented cells mixedwith 100 CCID₅₀ (tissue culture infectious doses) of HIV-1 IIIB, whichlysed 100% of the MT-2 cells. The cytopathic effect ("CPE") of thecompounds on uninfected cells was measured using the same methodology.

Pharmaceutical excipients, except compounds insoluble in water,detergents and oxidizing agents, were screened for anti-HIV-1 activityby an assay measuring fusion between infected and uninfected cells. Onlytwo compounds, cellulose acetate phthalate (CAP) and hydroxypropylmethylcellulose phthalate (HPMCP), had inhibitory activity. Anotherpolymeric phthalate, vinyl acetate phthalate, had no inhibitoryactivity. Other cellulose derivatives, for example,carboxymethylcellulose, also lacked activity. The two selectedcompounds, CAP and HPMCP, also inhibited HIV-1 infection, as measured byinhibition of the cpe and of the production of the HIV-1 nucleocapsidantigen p24 (Table 1B). The two compounds were not toxic for uninfectedcells at concentrations ≧2,500 μg/ml. The anti-HIV-1 activity of CAP wasbetter than that of HPMCP, but both compounds provided desirableresults.

Since herpesviruses are also frequently transmitted sexually, it was ofinterest to determine whether CAP might also inhibit infection by theseviruses. Herpesvirus type 1 (HSV-1) and type 2 (HSV-2) were selected totest this possibility. Results summarized in FIG. 1 indicate that CADinhibited infection by both HSV-1 and HSV-2. Similarly, CAD inhibitedinfection by HCMV. With respect of FIG. 1, virus preparations weretested for infectivity using a readout system based on the quantitationof β-galactosidase (absorbance at 410 nm).

                  TABLE 1A                                                        ______________________________________                                        Pharmaceutical Excipients Tested for Anti-HIV-1 Activity                                          Inhibition of Cell Fusion                                                     *ED.sub.50 ± SD                                        COMPOUND            (μg/ml)                                                ______________________________________                                        Acacia               --**                                                     Acesulfame Potassium                                                                              --                                                        Alginic Acid        --                                                        Ascorbyl Palmitate  --                                                        Aspartame           --                                                        Benzyl Benzoate     --                                                        Bronopol            --                                                        Butylated Hydroxyanisole                                                                           --                                                       Butylated Hydroxytoluene                                                                           --                                                       Butylparaben        --                                                        Carbomer 934P       --                                                        Carboxymethylcellulose                                                                            --                                                        Cellulose Acetate Phthalate                                                                       51.91 ± 1.32                                           Chlorocresol        --                                                        Croscarmellose Sodium                                                                             --                                                        Dextrates           --                                                        Dibutyl Sebacate    --                                                        Ethylparaben        --                                                        Hydroxyethyl Cellulose                                                                            --                                                        Hydroxypropyl Cellulose                                                                           --                                                        Hydroxypropyl Methylcellulose                                                                     --                                                        Hydroxypropyl Methylcellulose Phthalate                                                           68.30 ± 11.48                                          Imidurea            --                                                        Maltodextrin        --                                                        Maltol              --                                                        Menthol             --                                                        Methylcellulose     --                                                        Methylparaben       --                                                        Poloxamer           --                                                        Polymethacrylates   --                                                        Povidone            --                                                        Propyl Gallate      --                                                        Propylene Carbonate --                                                        Propylene Glycol Alginate                                                                         --                                                        Propylparaben       --                                                        Saccharin           --                                                        Sodium Alginate     --                                                        Sodium Cyclamate    --                                                        Sodium Starch Glycolate                                                                           --                                                        Sodium Stearyl Fumarate                                                                           --                                                        Sorbic Acid         --                                                        Pregelatinized Starch                                                                             --                                                        Triacetin           --                                                        Vanillin            --                                                        Vinylacetate Phthalate                                                                            --                                                        Xanthan Gum         --                                                        ______________________________________                                         *ED.sub.50 = Effective dose for 50% inhibition of HIV1 induced cell           fusion.                                                                       **-- = means no inhibitory activity on HIV1 induced cell fusion at the        final concentration of 1000 μg/ml.                                    

                  TABLE 1B                                                        ______________________________________                                        Pharmaceutical Excipients Tested for Anti-HIV-1 Activity                                              Hydroxypropyl                                                 Cellulose       Methylcellulose                                       Inhibition                                                                            Acetate Phthalate                                                                             Phthalate                                             of HIV-1                                                                              ED.sub.50 * ± SD                                                                     ED.sub.90 * ± SD                                                                     ED.sub.50 * ± SD                                                                   ED.sub.90 * ± SD                       Infection                                                                             (μg/ml)                                                                              (μg/ml)                                                                              (μg/ml)                                                                            (μg/ml)                                ______________________________________                                        P24     2.54 ± 4.76 ± 4.76 ±                                                                             8.86 ±                                 Production                                                                            0.16      1.05      1.20    1.11                                      CPE     3.68 ± 7.62 ± 7.79 ±                                                                             15.62 ±                                        0.74      1.66      1.30    7.61                                      Cell Fusion                                                                           51.91 ±                                                                              94.89 ±                                                                              68.30 ±                                                                            157.32 ±                                       1.32      3.12      11.48   32.86                                     ______________________________________                                         *ED.sub.50(90) = Effective dose(s) for 50% (90%) inhibition of HIV1           mediated p24 production, CPE and cell fusion.                            

                  TABLE 2                                                         ______________________________________                                        Compounds Known Not to Have Anti-HIV-1 Activity                               ______________________________________                                                  Albumin                                                                       Alpha Tocopherol                                                              Ascorbic Acid                                                                 Benzoic Acid                                                                  Benzyl Alcohol                                                                Dibasic Calcium Phosphate                                                     Calcium Sulfate                                                               Cholesterol                                                                   Citric Acid Monohydrate                                                       Cyclodextrins                                                                 Dextrin                                                                       Dextrose                                                                      Diethanolamine                                                                Diethyl Phthalate                                                             Edetic Acid                                                                   Ethyl Maltol                                                                  Ethyl Vanillin                                                                Fructose                                                                      Fumaric Acid                                                                  Gelatin                                                                       Liquid Glucose                                                                Glycerin                                                                      Guar Gum                                                                      Lactic Acid                                                                   Lactose                                                                       Malic Acid                                                                    Maltitol Solution                                                             Mannitol                                                                      Meglumine                                                                     Monoethanolamine                                                              Polyethylene Glycol                                                           Polyvinyl Alcohol                                                             Potassium Chloride                                                            Potassium Citrate                                                             Potassium Sorbate                                                             Propylene Glycol                                                              Sodium Bicarbonate                                                            Sodium Chloride                                                               Sodium Citrate Dihydrate                                                      Sodium Metabisulfite                                                          Dibasic Sodium                                                                Monobasic Sodium                                                              Sodium Propionate                                                             Sorbitol                                                                      Starch                                                                        Sterilizable Maize Starch                                                     Sucrose                                                                       Compressible Sugar                                                            Confectioner's Sugar                                                          Sugar Spheres                                                                 Tartaric Acid                                                                 Thimerosal                                                                    Triethanolamine                                                               Triethyl Citrate                                                              Xylitol                                                             ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Compounds Insoluble in Water or Buffers                                       ______________________________________                                                Bentonite                                                                     Calcium Carbonate                                                             Calcium Stearate                                                              Carboxymethylcellulose Calcium                                                Microcrystalline Cellulose                                                    Powdered Cellulose                                                            Cetostearyl Alcohol                                                           Cetyl Alcohol                                                                 Cetyl Esters Wax                                                              Crospovidone                                                                  Ethylcellulose                                                                Kaolin                                                                        Magnesium Aluminum Silicate                                                   Magnesium Carbonate                                                           Magnesium Oxide                                                               Magnesium Stearate                                                            Magnesium Trisilicate                                                         Polacrilin Potassium                                                          Shellac                                                                       Colloidal Silicon Dioxide                                                     Suppository Bases                                                             Talc                                                                          Titanium Dioxide                                                              Tragacanth                                                                    Zein                                                                          Zinc Stearate                                                         ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Organic Compounds, Oils, Waxes and Solvents and Detergents                    Solubilizing Cell Membranes and Envelopes of Lipid Containing                 ______________________________________                                        Viruses                                                                       Alcohol                                                                       Benzalkonium Chloride                                                         Benzethonium Chloride                                                         Canola Oil                                                                    Hydrogenated Castor Oil                                                       Cetrimide                                                                     Corn Oil                                                                      Cottonseed Oil                                                                Docusate Sodium                                                               Ethyl Oleate                                                                  Glyceryl Monooleate                                                           Glyceryl Monostearate                                                         Glyceryl Palmitostearate                                                      Glycofurol                                                                    Isopropyl Alcohol                                                             Isopropyl Myristate                                                           Isopropyl Palmitate                                                           Lanolin                                                                       Lanolin Alcohols                                                              Hydrous Lanolin                                                               Lecithin                                                                      Medium Chain Triglycerides                                                    Mineral Oil                                                                   Light Mineral Oil                                                             Mineral Oil and Lanolin Alcohols                                              Oleic Acid                                                                    Paraffin                                                                      Peanut Oil                                                                    Petrolatum                                                                    Petrolatum and Lanolin Alcohols                                               Polyoxyethylene Alkyl Ethers                                                  Polyoxyethylene Castor Oil Derivatives                                        Polyoxyethylene Sorbitan Fatty Acid Esters                                    Polyoxyethylene Stearates                                                     Sesame Oil                                                                    Sodium Lauryl Sulfate                                                         Sorbitan Esters (Sorbitan Fatty Acid Esters)                                  Soybean Oil                                                                   Stearic Acid                                                                  Stearyl Alcohol                                                               Hydrogenated Vegetable Oil Type 1                                             Anionic Emulsifying Wax                                                       Carnauba Wax                                                                  Microcrystalline Wax                                                          Nonionic Emulsifying Wax                                                      White Wax                                                                     Yellow Wax                                                                    ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Gases Used for Example in Aerosol Propellants                                 ______________________________________                                                 Butane                                                                        Carbon Dioxide                                                                Chlorodifluoroethane                                                          Chlorodifluoromethane                                                         Dichlorodifluoromethane                                                       Dichlorotetrafluoroethane                                                     Difluoroethane                                                                Dimethyl Ether                                                                Isobutane                                                                     Nitrogen                                                                      Nitrous Oxide                                                                 Propane                                                                       Tetrafluoroethane                                                             Trichloromonofluoromethane                                           ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Oxidizing Agents and Disinfectants                                            ______________________________________                                        Chlorhexidine                                                                             Phenol        Phenylmercuric Acetate                              Chlorobutanol                                                                             Phenoxyethanol                                                                              Phenylmercuric Borate                               Cresol      Phenylethyl Alcohol                                                                         Phenylmercuric Nitrate                              ______________________________________                                    

Example 2:

Formulations of CAP

The formulation of CAP and HPMCP for topical vaginal application as anantiviral agent or virucide to prevent the sexual transmission of HIV-1and herpesviruses, respectively, represented a difficult challenge whichcould be overcome only by innovative approaches. Both CAP and HPMCP areinsoluble in water and can be solubilized in water by adjusting the pHof the environment to ≈6 or above (Handbook of PharmaceuticalExcipients, 2nd Edition, edited by Ainley Wade and Paul J. Weller,American Pharmaceutical Association, Washington, (1994)), or by the useof appropriate organic solvents. On the other hand, vaginal secretionsfrom healthy, reproductive-age women are characteristically acidic (pHvalues of 3.4 to 6.0) (S. Voeller, D. J. Anderson, "HeterosexualTransmission of HIV", JAMA, 267, 1917-1918, (1992)). Consequently, thetopical application of a formulation in which either CAP or HPMCP wouldbe soluble (i.e., pH=≧6) would be expected to contribute to a vaginalenvironment which is physiologically undesirable. Nevertheless, attemptswere made to formulate CAP or HPMCP in gels/creams which are customarilyused for vaginal applications as moisturizers and/or contraceptiveagents. These included the following: hydroxyethylcellulose gels (e.g.,K-Y JELLY, Johnson and Johnson, Raritan, N.J.); carbomer 934P based gels(e.g., REPLENS, Roberts Pharmaceuticals, Inc., Mississauga, Ontario,Canada; Taro gel, Taro Pharmaceuticals, Inc., Bramalea, Ontario,Canada); hydroxypropyl methylcellulose and carbomer 934P based gels(e.g., H-R lubricating jelly, Carter-Wallace, Inc., New York, N.Y.);polyglyceryl methacrylate (Gyne-Moistrin Moisturizing Gel(Shering-Plough Healthcare Products, Inc., Mississauga, Ontario,Canada)), and gels containing carbomer 934P and hydroxypropylmethylcellulose alone. All the aforementioned formulations have water astheir major constituent. When the preparations of CAP and HPMCP in theabove gels were submitted to accelerated stability studies for 7 days at45° C. and subsequently tested for anti-HIV-1 activity, no antiviralactivity was detected. This was probably due to the hydrolysis of eachof these cellulose derivatives, resulting in the release of acetic andphthalic acids and leading to diminished anti-HIV-1 activity.

To avoid this problem, it was decided to dissolve the cellulosederivatives (experiments were carried out mostly with CAP which hashigher anti-HIV-1 activity in comparison with HPMCP; see Table 1Bherein) in organic solvents that are low in water content, yet watermiscible for in vivo compatibility, and nontoxic to vaginal mucosa,selected on the basis of preliminary studies. These solvents includedthe following: propylene glycol, propylene carbonate, benzyl alcohol,polyethylene glycol (PEG 400), dimethyl isosorbide, and ethoxydiglycol("TRANSCUTOL"). The solubility of CAP in these solvents ranges between5.3 to 30% (w/w). To increase the viscosity of these solutions, it wasnecessary to use them as gels/creams for topical applications. Eitherpolyvinyl pyrrolidone (PVP) and/or different poloxamers (e.g., PluronicF68) were added to the CAD solutions in the different organic solvents.To estimate the properties of the different formulations after contactwith a physiological environment, they were mixed with water orphysiological saline (0.14 M NaCl). Under these conditions, CAPprecipitated at the interface of the formulations with water (saline) inthe form of a large polymeric mass which would not be expected to haveantiviral activity and would not be appropriate for topical application.It was possible to overcome this problem by incorporating into the CAPcontaining formulation compounds which increase the pH upon contact withwater or a saline solution (=0.14 M NaCl), e.g., sodium acetate ortriethanolamine. Inclusion of the latter compounds into the formulationeliminated or diminished the problem of appearance of large CAPaggregates. Surprisingly, accelerated stability studies (incubation for7 days at 45° C.) of CAP in the above organic pharmaceuticalexcipients/solvents, containing in addition the aforementioned gellingand buffering agents, resulted in complete loss of anti-HIV-1 activity.This activity was retained if the buffering agents were omitted andadded only before initiating the assay for anti-HIV-1 activity. Thus, insummary, the CAP formulations in organic solvents containing also abuffering agent represent formulations unsuitable for topicalapplications, either because the active ingredient, CAP, precipitatesfrom the formulation in a large polymeric mass upon contact withphysiological fluids (in the absence of appropriate buffering agentsincorporated into the formulation) or are converted into uselessformulations lacking anti-HIV-1 activity, because of inactivation of theactive ingredient, CAP.

To avoid the above problems, the possibility of using CAP in the form ofa micronized preparation in suspension was explored. This necessitatedthe use of a solvent in which CAP would not be soluble, since otherwisethe results obtained would be expected to be exactly the same as werethose referred to above. A solvent with such properties is actuallywater in which neither CAP nor HPMCP are soluble (Handbook ofPharmaceutical Excipients, 2nd Edition, edited by Ainley Wade and PaulJ. Weller, American Pharmaceutical Association, Washington (1994)).

A formulation containing water and a commercially available micronizedform of CAP ("AQUATERIC" from the FMC Corporation, Philadelphia, Pa.),containing in addition to CAP (63 to 70 wt %), poloxamer and distilledacetylated monoglycerides, was prepared. Thickening agents, i.e., PVPand/or pluronic F68, were added to the water suspension of the"AQUATERIC". When this gel was submitted to accelerated stabilitystudies (7 days at 45° C.) and then tested for anti-HIV-1 activity,essentially no antiviral activity was recovered. Thus, another solventwas needed in which the CAP ("AQUATERIC") would not be soluble and wouldnot lose antiviral activity. Surprisingly, glycerol (very similar topropylene glycol in which CAP is soluble to ≈30% w/w) meets both theserequirements. Based on this discovery, a formulation of CAP("AQUATERIC") was prepared as follows: 200 mg of PVP (MW 40,000,Spectrum) were dissolved in 1 ml of glycerol. Subsequently, 50 mg ofCrospovidone (Polyplasdone INF-10, ISP Technologies) were suspended inthe solution followed by the addition of 286 mg of "AQUATERIC". The PVPand Crospovidone were added to prevent the separation of the "AQUATERIC"microsuspension from glycerol. The resulting formulation maintained itsuniformity over time and also its anti-HIV-1 activity following anaccelerated stability test carried out under conditions described above.

In summary, the following properties of CAD dictated its formulation:(a) low solubility in aqueous solutions at pH<6, (b) hydrolysis duringstorage in aqueous solutions at room temperature, and (c) solubility inseveral biocompatible organic solvents; for example, propylenecarbonate, propylene glycol and polyethylene glycol, from which CAPprecipitates upon contact with aqueous solvents. To avoid theseproblems, a formulation of micronized CAP in glycerol (in which CAP isnot soluble), "CAP Formulation I", was prepared.

"CAP Formulation I" is a preparation of micronized CAP ("AQUATERIC"containing 66-73% of CAP, a polyoxyethylene-polyoxypropylene blockcopolymer and distilled acetylated monoglycerides, used in aqueous mediaas an enteric film-coating liquid (FMC Corporation, Philadelphia, Pa.)),(15.9 g) was mixed with glycerol (70.2 g) and polyvinylpyrrolidone K-30(Spectrum Quality Products, Inc., New Brunswick, N.J.) (11.1 g) andCrospovidone NF (ISP Technologies, Inc., Wayne, N.J.) (2.8 g) were addedin order to maintain the micronized CAP in suspension.

Another formulation, "CAP Formulation II", was prepared by replacingpolyvinylpyrrolidone+Crospovidone with colloidal silicon dioxide M-5P(Cabot Corp., Cab-O-Sil Division, Tuscola, Ill.), an excipient with anestablished use in vaginal preparations. CAP Formulation II contained23.7 g "AQUATERIC" and 7.89 g silica per 100 g glycerol.

All components of these two CAP Formulations were USP grade and havebeen approved for human medicinal use.

Example 3

Measurement of Inhibitory Ability Against HSV-1 and HSV-2 and AgainstHCMV

The following method was used to measure the inhibitory activity: 500 μlof compounds (at distinct dosages) in Eagle's Minimum Essential Medium(EMEM) were mixed with an equal volume of appropriately dilutedinfectious HSV-1 or HSV-2. The mixture was added to ELVIS HSV cells in24-well plates. The ELVIS cells as well as the media were provided byDiagnostic Hybrids, Inc. (Athens, Ohio).

ELVIS cells are derived by selection of G400-resistant coloniesfollowing cotransfection of baby hamster kidney cells with a plasmidwhich contains a G418-antibiotic-resistant marker and a plasmid whichcontains an Escherichia coli LacZ gene placed behind an inducible HSVpromoter. The promoter is from HSV-1 UL39 which encodes ICP6, the largesubunit of ribonucleotide reductase (RR1). This promoter has a number offeatures which make it ideal for the detection of HSV. First, there isno constitutive expression from this promoter in uninfected cells.Second, activation of the promoter appears to be specific for HSV.Third, expression from this promoter occurs within hours afterinfection. Fourth, this promoter is strongly transactivated by thevirion associated trans-activator protein VP16. As early as six hoursafter infection, HSV-infected cells can be detected by histochemicalstaining for β-galactosidase activity (Stabell E. C. and Olivo P. D.,"Isolation of a Cell Line for Rapid and Sensitive Histochemical Assayfor the Detection of Herpes Simplex Virus", J. Virological Methods, 38,195-204, (1992)).

Twenty four hours after HSV infection, in the presence and absence ofgraded quantities of the test compounds, the ELVIS cells were lysed withTRITON X-100 and β-galactosidase in the cell lysates was determined byan ELISA kit provided by Five Prime→Three Prime, Inc. (Boulder, Colo.).This ELISA kit is capable of detecting and quantitating picogram levelsof E. coli β-galactosidase protein expressed in transformed bacteria oreukaryotic cells and tissues. The method is based on detection of theβ-galactosidase protein rather than on the enzymatic activity.β-galactosidase from E. coli is a tetrameric enzyme composed of fouridentical subunits. The individual subunits do not exhibit enzymeactivity and therefore are not detectable by standard enzyme activityassays. The Five Prime→Three Prime β-galactosidase ELISA kit overcomesthis limitation by detecting the actual protein that is expressed.

The surprising conclusion of the described experiments was thatcellulose acetate phthalate and hydroxypropyl methylcellulose phthalate,of all the excipients tested, were unique in having potent antiviralactivity against both HIV-1 and HSV-1 and HSV-2, and against otherviruses belonging to the herpesvirus group. Alternately, antiviralactivity against HSV-1 was measured using HSV vgCL5, a recombinant virusin which the expression of β-galactosidase ("β-gal") is under thecontrol of the HSV-1 late gene gC regulatory region (Weir, J. P.,Steffy, K. R., Sethna, M., "An Insertion Vector for the Analysis of GeneExpression During Herpes Simplex Virus Infection", Gene 91990), 89,271-274).

In this test, 50 μl of MEM tissue culture medium containing 5% FBS andgraded concentrations of CAP was mixed with 100 μl of Vero cells in thesame FBS-containing medium (10⁶ cells/ml) for 30 minutes at 25° C.Subsequently, 50 μl of HSV vgCL5, at a dilution sufficient to infectapproximately 50% of cells, as determined by in situ staining forβ-galactosidase, was added and the mixture was placed into wells of96-well plates. After incubation at 37° C. for 24 hours, the cells wereeither immediately lysed or frozen for 1 to 5 days for storage andsubsequently lysed by adding to the cells and medium, 50 μl of 2.5%(v/v) TRITON X-100, containing protease inhibitors (PMSF, leupeptin andpepstatin, all at 10 μg/ml). β-galactosidase in the lysed preparationswas detected by the ELISA kit from Five Prime→Three Prime.

Inhibitory activity against human cytomegalovirus (HCMV) was measuredusing the HCMV strain RC-256 (ATCC VR-2536), a recombinant of HSMV Townecontaining the E.coli LacZ gene (Spaete, R. R., Mocarski, E. S.,"Regulation of Cytomegalovirus Gene Expression: α and β Promoters AreTrans Activated by Viral Functions in Permissive Human Fibroblasts", J.Virol., (1985), 56, 135-143).

The inhibitory effect on CMV on replication of the β-galactosidaseexpressing HCMV RC-256 was measured under conditions similar to thosedescribed for HSV vgCL5, except that the cell-virus mixtures were keptfor 48 hours at 37° C. before measuring β-galactosidase levels and humanforeskin fibroblast (HFF) cells were used.

Example 4

Virucidal Activity of CAP Formulations

Both the anti-HIV-1 activity and the anti-HSV-1 and anti-HSV-2activities of the CAP formulation, before or after stability testing,corresponded to the content of CAP in the preparation and to resultsshown in Table 1B and FIGS. 1 and 2 for HIV-1 and herpesviruses,respectively.

When a preparation of HIV-1 was mixed 1:1 with either an equal volume ofa suspension of "AQUATERIC" in glycerol or with an equal volume of theabove-mentioned formulation for 5 minutes at 37° C., a complete loss ofHIV-1 infectivity occurred (FIG. 4). The inactivation of HIV-1infectivity can be ascribed to the complete disruption of HIV-1 virions,as demonstrated by the quantitative release of the internal nucleocapsidantigen p24 (FIG. 3). Similarly, the infectivity of both HSV-1 and HSV-2was destroyed by suspensions of "AQUATERIC" in glycerol or the"AQUATERIC"-glycerol formulation with PVP and Crospovidone (FIGS. 5 and6).

With respect of FIG. 3, serial dilutions of untreated and treated HIV-1were tested for p24 by ELISA. As a positive control, HIV-1 treated withthe detergent NP40 was also tested. The results obtained with the"AQUATERIC"-glycerol formulation containing PVP and Crospovidone andN-40 were identical and indicated an 100-fold increase of released p24antigen, as compared to background levels corresponding to untreatedvirus. The infectivity of HIV-1 was also eliminated by treatment withthe "AQUATERIC"-glycerol formulation containing PVP and Crospovidone.

Concerning FIG. 5, serial dilutions of the virus preparations before orafter treatment with "AQUATERIC" were tested for infectivity using twodistinct readout systems based on quantitation of β-galactosidase(absorbance at 410 nm).

Regarding FIG. 6, virus preparations were mixed 1:1 with an"AQUATERIC"-glycerol formulation with PVP and Crospovidone for 5 minutesat 37° C. Serial dilutions of the virus preparations were tested forinfectivity using a readout system based on quantitation ofβ-galactosidase (absorbance at 410 nm).

CAP as an acetic acid ester of cellulose may undergo hydrolysis inaqueous environments. Therefore, CAP formulations in organic solventswere explored. The best results were obtained by dispersing micronizedCAP, containing in addition to CAP a polyoxyethylene-polyoxypropyleneblock copolymer and distilled acetylated monoglycerides ("AQUATERIC"),in glycerol to which povidone and crospovidone were added to stabilizethe suspension (CAP Formulation I). An alternative, CAP Formulation IIcontaining colloidal silicone dioxide, instead of povidone andcrospovidone, was also prepared. Mixing of these formulations with anequal volume of a suspension of infectious HIV-1, it was possible toshow that this treatment resulted in disintegration of HIV-1 particlesmeasurable by the release of the nucleocapsid antigen p24 (FIG. 3). TheCAP Formulation was as effective as the detergent NP40 in releasing thenucleocapsid antigen from virus particles. Similar results were obtainedwhen seminal fluid or whole blood were added to the HIV-CAP formulationmixtures (FIG. 7).

The CAP Formulations also inactivated the infectivity of HSV-1 and HSV-2(FIG. 6). Similar results were obtained using HCMV (FIG. 8). Addition ofseminal fluid or whole blood to the CAP formulation did not interferewith its virus-inactivating properties under the conditions used,indicating that the microbicidal potential of CAP will be maintainedunder analogous conditions in vivo.

Example 5

Inactivation of Several Sexually Transmitted Pathogens by CAPFormulations

To study the effect of formulated CAD on HIV-1, HSV-1, HSV-2 and HCMV,the respective virus suspensions were mixed with an equal volume of theCAP Formulations I and II (each pre-warmed at 37° C.). After incubationfor 5 minutes at 37° C., the suspensions were filtered through a 0.45μfilter and subsequently centrifuged to remove suspended CAP. Thesupernatant fluids were tested for virus infectivity. This separationcould not be accomplished using CAP Formulation II, and thecorresponding virus-CAP formulation mixtures were tested directly forvirus infectivity. In control experiments, formulations lacking CAP("AQUATERIC"), mixed with respective virus preparations and PBS (0.14NaCl, 0.01 M phosphate pH 7.2) mixed with virus were used, respectively.Serial two-fold dilutions of the respective virus preparations weretested for HIV-1 and herpesvirus infectivity using β-galactosidasereadout systems for herpesviruses, as described above. In someexperiments, seminal fluid (0.6 ml; provided by New England ImmunologyAssociates, Cambridge, Mass.) or whole human heparinized blood (0.11 ml)were added to the CAP Formulations (1 ml), followed by addition of virus(1 ml), in order to evaluate their effect on virus inactivation.

To determine whether or not the CAP Formulations affect the integrity ofHIV-1, a preparation of purified virus (strain IIIB; 5 μl; 1.52×10¹⁰virus particles/ml; Advanced Biotechnologies, Columbia, Md.) was mixedwith 45 μl of 0.14 M NaCl and 50 μl of the respective CAP Formulation,each prewarmed to 37° C. After 5 minutes at 37° C., 400 μl of 0.14 MNaCl were added, and the mixture was filtered through a 0.45μ filterprewashed with 0.14 M NaCl, 0.01 M Tris pH 7.2, 0.02 NaN₃ (TS). Serial5-fold dilutions of the filtrate were tested by ELISA for the p24antigen, as described above. In control experiments, either untreatedHIV-1 IIIB or virus treated with the detergent NP40 (final concentration5 mg/ml; 5 minutes at 37° C.) were equally diluted and tested for thep24 antigen. Similar experiments were done with the CAP Formulations inthe presence of seminal fluid or whole human blood (proportions givenabove). When whole blood was added to the CAP Formulations, the dilutedvirus preparations were centrifuged at 2000×g for 5 minutes beforefiltration through 0.45μ filters.

Example 6

Inactivation of Bacterial Pathogens

To test the effect of the CAP Formulation I (a preparation of micronizedCAP ("AQUATERIC" containing 66-73% of CAP, apolyoxyethylene-polyoxypropylene block copolymer and distilledacetylated monoglycerides, used in aqueous media as an entericfilm-coating liquid (FMC Corporation, Philadelphia, Pa.)), (15.9 g) wasmixed with glycerol (70.2 g) and polyvinylpyrrolidone K-30 (SpectrumQuality Products, Inc., New Brunswick, N.J.) (11.1 g) and CrospovidoneNF (ISP Technologies, Inc., Wayne, N.J.) (2.8 g) were added in order tomaintain the micronized CAP in suspension) on distinct bacterialsexually transmitted disease (STD) pathogens and Lactobacilli,respectively, equal volumes of the CAP Formulation and a suspension ofbacteria (10⁸ to 10⁹ cells/ml in 0.14 M NaCl) were mixed and incubatedat 37° C. for either 5 or 15 minutes. Subsequently, 50 μl of 0.43 M Na₃PO₄.12H₂ O were added per 1 ml of the suspension. After 5 minutes atroom temperature and intermittent mixing, the neutralized suspensionswere serially diluted 10-fold in PBS or appropriate broth media. A 20 μlvolume of each dilution was inoculated per well of a microtiter platecontaining growth media or onto the appropriate agar plate and incubatedunder appropriate conditions to monitor bacterial growth. Included witheach experiment was a positive antibiotic control to demonstrateinhibition of bacterial growth and a control without the formulation orantibiotic to monitor the growth of the bacteria.

The bacterial strains, the corresponding growth media and the controlantibiotics used were: Lactobacillus crispatus, ATCC 33820(lactobacillus broth; tetracycline 0.5 to 64 μg/ml); Neisseriagonorroheae, ATCC 49226 (GC media [Becton Dickinson MicrobiologySystems, Sparks, Md.) supplemented with 500 μl of a 2% hemoglobin stockand 10 ml of "ISOVITALEX" (Becton Dickinson Microbiology Systems,Sparks, Md.) per liter of media; Ceftriaxone at 0.005 to 0.128 μg/ml);Haemophilus ducreyi, ATCC 33940 (Revised Ducreyi Medium, American TypeCulture Collection (ATCC) Culture Medium 1724) supplemented with 10% FBS(Life Technologies, Gaithersburg, Md.) and 1% "ISOVITALEX" (BectonDickinson Microbiology Systems, Sparks, Md.); Tetracycline 0.125 to 64μg/ml; Trichomonas vaginalis, ATCC 30092 (Modified Fuji media (OhkawaM., Yamaguchi, K., Tokunaga, S. et al., "The Incidence of TrichomonasVaginalis in Chromic Prostatis Patients Determined by Culture Using aNewly Modified Liquid Medium", J. Infect. Dis., (1992), 166, 1205-1206);metronidazole 1 to 128 μg/ml).

The guidelines established by the Environmental Protection Agency(Efficacy Data Requirements: Virucides DISITSS-7, Nov. 12, 1981,Modifying AOAC Methods 4.007-4.014, Office of Pesticide Research,Environmental Protection Agency, Washington, D.C.) were followed tostudy the effect of CAP Formulation I on Chlamydia trachomatis (strainLGV type III; ATCC VR-903). After incubation with the formulation andneutralization, the preparation was diluted in DMEM growth mediacontaining 3.6 mM L-glutamine, 45 μg/ml gentamicin and 8.9% FBS, andserial 10-fold dilutions were prepared. The dilutions were added toMcCoy cell monolayers (ATCC CRL 1696) and the infection was determinedby microscopic observation of cpe, or by staining of inclusion orelementary bodies by iodine staining after 7 days incubation at 37° C.with 55 CO₂.

Thus, the above described CAP Formulation I was tested for itsinactivating effect on the following sexually transmitted pathogens:Chlamydia trachomatis, Trichomonas vaginalis, Neisseria gonorrhoeae,Haemophilus ducreyi, and on Lactobacillus crispatus. The results shownin the following Table 7 indicate that all bacteria, exceptLactobacillus crispatus, lost the capacity to replicate after exposureto the CAP Formulation. The activity of the CAP Formulation againstTreponema palladium was not tested because the unavailability ofappropriate in vitro assays. Thus, the CAP Formulation was activeagainst four major non-viral STD pathogens, but did not affectLactobacilli, an essential component of the normal vaginal flora.

                                      TABLE 7                                     __________________________________________________________________________    Antibacterial Activity of Formulated CAP                                      Trichomonas vaginalis                                                                          Neisseris gonorrhoeae                                                                     Haemophilus ducreyl                                                                       Lactobacillus crispatus                                                                   Chiamydis                                                                     trachomatis                       5 Min                                                                             15 Min  5 Min                                                                             15 Min  5 Min                                                                             15 Min  5 Min                                                                             15 Min   10 Min                   Growth                                                                            Expo-                                                                             Expo-                                                                             Growth                                                                            Expo-                                                                             Expo-                                                                             Growth                                                                            Expo-                                                                             Expo-                                                                             Growth                                                                            Expo-                                                                             Expo-                                                                             Growth                                                                             Expo-               Dilution                                                                           Control                                                                           sure                                                                              sure                                                                              Control                                                                           sure                                                                              sure                                                                              Control                                                                           sure                                                                              sure                                                                              Control                                                                           sure                                                                              sure                                                                              Control                                                                            sure                __________________________________________________________________________    10.sup.0                                                                           nd  nd  nd  +   0   0   +   +   0   5+  5+  5+                           10.sup.-1                                                                          +   0   0   +   0   0   +   +   +/- 5+  5+  5+                           10.sup.-2                                                                          +   0   0   +   0   0   +   +   0   5+  5+  3+  4+   0                   10.sup.-3                                                                          +   0   0   +   0   0   +   +   0   3+  4+  2+  4+   0                   10.sup.-4                                                                          +   0   0   +   0   0   +   +   0   2+  3+  2+  4+   0                   10.sup.-5                                                                          0   0   0   +   0   0   +   +/- 0   2+  2+  1+  0    0                   10.sup.-6                                                                          0   0   0   0   0   0   +   +/- 0   2+  1+  1+  0    0                   10.sup.-7                                                                          0   0   0   0   0   0   +   +/- 0   0   1+  0   0    0                   __________________________________________________________________________     nd = not done                                                                 "0" = No growth                                                               "+" = Growth                                                                  "+/-" = Slight Growth                                                         "1+" = Trace growth                                                           "2+" = Light growth                                                           "3+" = Moderate growth                                                        "4+" = Moderately Dense Growth                                                "5+" = Dense Growth                                                      

It will be appreciated that the instant specification is set forth byway of illustration and not limitation, and that various modificationsand changes may be made without departure from the spirit and scope ofthe present invention.

What is claimed is:
 1. A method for decreasing the frequency oftransmission of human cytomegalovirus, comprising administering to ahuman an effective anti-cytomegalovirus amount of at least one cellulosephthalate selected from the group consisting of cellulose acetatephthalate and hydroxypropyl methylcellulose phthalate, either alone orin combination with a pharmaceutically acceptable carrier or diluent. 2.A pharmaceutical composition for decreasing the frequency oftransmission of a virus selected from the group consisting of humanimmunodeficiency virus and herpesvirus, or for preventing thetransmission of or for treating a sexually transmitted bacterialinfection comprising an effective anti-human immunodeficiency virusamount or anti-herpesvirus amount or an effective anti-bacterial amountof a composition comprising (a) 1 to 25 wt. % of a micronized celluloseacetate phthalate, (b) glycerol or squalane, (c) polyvinylpyrrolidone,and (d) cross-linked 1-ethenyl-2-pyrrolidinone homopolymer, wherein theglycerol or squalane, polyvinylpyrrolidone and cross-lined1-ethenyl-2-pyrrolidinone homopolymer are in amounts such that thecomposition results in a homogeneous cream.
 3. A pharmaceuticalcomposition for decreasing the frequency of transmission of a virusselected from the group consisting of human immunodeficiency virus andherpesvirus, or for preventing the transmission of or for treating asexually transmitted bacterial infection comprising an effectiveanti-human immunodeficiency virus amount or anti-herpesvirus amount oranti-bacterial amount of a composition comprising (a) 1 to 25 wt. % of amicronized cellulose acetate phthalate, (b) glycerol or squalane, and(c) colloidal silicon dioxide, wherein the glycerol or squalane andcolloidal silicon dioxide are in amounts such that the compositionresults in a homogeneous cream.
 4. A pharmaceutical composition fordecreasing the frequency of transmission of a virus selected from thegroup consisting of human immunodeficiency virus and herpesvirus, or forpreventing the transmission of or for treating a sexually transmittedbacterial infection comprising an effective anti-human immunodeficiencyvirus amount or anti-herpesvirus amount or anti-bacterial amount of acomposition comprising (a) micronized cellulose acetate phthalate, (b)glycerol or squalane, and (c) colloidal silicon dioxide and/orpolyvinylpyrrolidone and cross-linked 1-ethenyl-2-pyrrolidinonehomopolymer, wherein the components (a), (b) and (c) are in amounts suchthat the composition results in a semi-solid dough or putty.
 5. Thepharmaceutical composition of claim 3, wherein the micronized celluloseacetate phthalate is prepared by dissolving cellulose acetate phthalateand polyvinylpyrrolidone in dimethyl sulfoxide and then adding waterwhile mixing to form a fine precipitate of cellulose acetate phthalatecontaining polyvinylpyrrolidone, washing with water and thenfreeze-drying the resultant powder.
 6. The pharmaceutical composition ofclaim 4, wherein the micronized cellulose acetate phthalate is preparedby dissolving cellulose acetate phthalate and polyvinylpyrrolidone indimethyl sulfoxide and then adding water while mixing to form a fineprecipitate of cellulose acetate phthalate containingpolyvinylpyrrolidone, washing with water and then freeze-drying theresultant powder.
 7. The pharmaceutical composition of claim 2, wherein(b) is glycerol.
 8. The pharmaceutical composition of claim 2, wherein(b) is squalane.
 9. The pharmaceutical composition of claim 3, wherein(b) is glycerol.
 10. The pharmaceutical composition of claim 3, wherein(b) is squalane.
 11. The pharmaceutical composition of claim 4, wherein(b) is glycerol.
 12. The pharmaceutical composition of claim 4, wherein(b) is squalane.
 13. The pharmaceutical composition of claim 11, wherein(c) is colloidal silicon dioxide.
 14. The pharmaceutical composition ofclaim 12, wherein (c) is colloidal silicon dioxide.
 15. Thepharmaceutical composition of claim 11, wherein (c) is colloidal silicondioxide, polyvinylpyrrolidone and cross-linked1-ethenyl-2-pyrrolidinone.
 16. The pharmaceutical composition of claim12, wherein (c) is colloidal silicon dioxide, polyvinylpyrrolidone andcross-linked 1-ethenyl-2-pyrrolidinone.
 17. A method for decreasing thefrequency of transmission of a virus selected from the group consistingof human immunodeficiency virus and herpesvirus, or for preventing thetransmission of or for treating a sexually transmitted bacterialinfection comprising topically administering to a human an effectiveanti-human immunodeficiency virus amount or anti-herpesvirus amount oran effective anti-bacterial amount of the pharmaceutical composition ofclaim
 2. 18. A method for decreasing the frequency of transmission of avirus selected from the group consisting of human immunodeficiency virusand herpesvirus, or for preventing the transmission of or for treating asexually transmitted bacterial infection comprising topicallyadministering to a human an effective anti-human immunodeficiency virusamount or anti-herpesvirus amount or an effective anti-bacterial amountof the pharmaceutical composition of claim
 3. 19. A method fordecreasing the frequency of transmission of a virus selected from thegroup consisting of human immunodeficiency virus and herpesvirus, or forpreventing the transmission of or for treating a sexually transmittedbacterial infection comprising topically administering to a human aneffective anti-human immunodeficiency virus amount or anti-herpesvirusamount or an effective anti-bacterial amount of the pharmaceuticalcomposition of claim
 4. 20. The method of claim 1, wherein the at leastone cellulose phthalate is cellulose acetate phthalate.
 21. The methodof claim 1, wherein the at least one cellulose phthalate ishydroxypropyl methylcellulose phthalate.
 22. The method of claim 1,wherein said cellulose phthalate is a combination of cellulose acetatephthalate and hydroxypropyl methylcellulose phthalate.
 23. The method ofclaim 17, wherein the virus is HIV-1.
 24. The method of claim 18,wherein the virus is HIV-1.
 25. The method of claim 19, wherein thevirus is HIV-1.
 26. The method of claim 17, wherein the virus is aherpesvirus.
 27. The method of claim 26, wherein the herpesvirus isHSV-1.
 28. The method of claim 26, wherein the herpesvirus is HSV-2. 29.The method of claim 18, wherein the virus is a herpesvirus.
 30. Themethod of claim 29, wherein the herpesvirus is HSV-1.
 31. The method ofclaim 29, wherein the herpesvirus is HSV-2.
 32. The method of claim 19,wherein the virus is a herpesvirus.
 33. The method of claim 32, whereinthe herpesvirus is HSV-1.
 34. The method of claim 32, wherein theherpesvirus is HSV-2.
 35. The method of claim 1, wherein theadministering is by topical administration.
 36. The pharmaceuticalcomposition of claim 2, wherein the micronized cellulose acetatephthalate is in an amount of 5 to 20 wt. %.
 37. The pharmaceuticalcomposition of claim 2, wherein the micronized cellulose acetatephthalate is in an amount of 12 to 18 wt. %.
 38. The pharmaceuticalcomposition of claim 3, wherein the micronized cellulose acetatephthalate is in an amount of 5 to 20 wt. %.
 39. The pharmaceuticalcomposition of claim 3, wherein the micronized cellulose acetatephthalate is in an amount of 12 to 18 wt. %.
 40. The pharmaceuticalcomposition of claim 2, wherein the micronized cellulose acetatephthalate is prepared by dissolving cellulose acetate phthalate andpolyvinylpyrrolidone in dimethyl sulfoxide and then adding water whilemixing to form a fine precipitate of cellulose acetate phthalatecontaining polyvinylpyrrolidone, washing with water and thenfreeze-drying the resultant powder.
 41. The pharmaceutical compositionof claim 3, wherein the micronized cellulose acetate phthalate is in anamount of 5 to 20 wt. %.
 42. The pharmaceutical composition of claim 3,wherein the micronized cellulose acetate phthalate is in an amount of 12to 18 wt. %.
 43. The pharmaceutical composition of claim 4, wherein themicronized cellulose acetate phthalate is in an amount of 5 to 20 wt. %.44. The pharmaceutical composition of claim 4, wherein the micronizedcellulose acetate phthalate is in an amount of 12 to 18 wt. %.
 45. Thepharmaceutical composition of claim 3, wherein the micronized celluloseacetate phthalate is prepared by dissolving cellulose acetate phthalateand polyvinylpyrrolidone in dimethyl sulfoxide and then adding waterwhile mixing to form a fine precipitate of cellulose acetate phthalatecontaining polyvinylpyrrolidone, washing with water and thenfreeze-drying the resultant powder.